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Determinants of lymphoid-myeloid lineage diversification

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Abstract and Figures

In recent years, investigators have made great progress in delineating developmental pathways of several lymphoid and myeloid lineages and in identifying transcription factors that establish and maintain their fate. However, the developmental branching points between these two large cell compartments are still controversial, and little is known about how their diversification is induced. Here, we give an overview of determinants that play a role at lymphoid-myeloid junctures, in particular transcription factors and cytokine receptors. Experiments showing that myeloid lineages can be reversibly reprogrammed into one another by transcription factor network perturbations are used to highlight key principles of lineage commitment. We also discuss experiments showing that lymphoid-to-myeloid but not myeloid-to-lymphoid conversions can be induced by the enforced expression of a single transcription factor. We close by proposing that this asymmetry is related to a higher complexity of transcription factor networks in lymphoid cells compared with myeloid cells, and we suggest that this feature must be considered when searching for mechanisms by which hematopoietic stem cells become committed to lymphoid lineages.
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ANRV270-IY24-23 ARI 23 February 2006 15:47
Determinants of
Lymphoid-Myeloid Lineage
Catherine V. Laiosa, Matthias Stadtfeld,
and Thomas Graf
Department of Developmental and Molecular Biology, Albert Einstein College
of Medicine, Bronx, New York 10461; email:
Annu. Rev. Immunol.
2006. 24:705–38
First published online as a
Review in Advance on
January 16, 2006
The Annual Review of
Immunology is online at
This article’s doi:
2006 by
Annual Reviews. All rights
Key Words
hematopoietic stem cell, hematopoietic lineage trees, transcription
factors, lineage commitment, lineage priming
In recent years, investigators have made great progress in delineating
developmental pathways of several lymphoid and myeloid lineages
and in identifying transcription factors that establish and maintain
their fate. However, the developmental branching points between
these two large cell compartments are still controversial, and little
is known about how their diversification is induced. Here, we give
an overview of determinants that play a role at lymphoid-myeloid
junctures, in particular transcription factors and cytokine receptors.
Experiments showing that myeloid lineages can be reversibly repro-
grammed into one another by transcription factor network perturba-
tions are used to highlight key principles of lineage commitment. We
also discuss experiments showing that lymphoid-to-myeloid but not
myeloid-to-lymphoid conversions can be induced by the enforced
expression of a single transcription factor. We close by proposing
that this asymmetry is related to a higher complexity of transcription
factor networks in lymphoid cells compared with myeloid cells, and
we suggest that this feature must be considered when searching for
mechanisms by which hematopoietic stem cells become committed
to lymphoid lineages.
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Lymphoid cells: B
and T lymphocytes
and their precursors;
also include natural
killer (NK) lineage
Myeloid cells: all
nonlymphoid cell
types, including
erythrocytes, and
(precursors of
stem cell (HSC):
cell capable of
differentiation into
all hematopoietic cell
lineages and of
CLP: common
lymphoid progenitor
CMP: common
myeloid progenitor
GMP: granulocyte-
Hematopoiesis has traditionally been studied
by two separate camps: immunologists pre-
dominantly involved with the development of
lymphoid cells such as B, T, and natural killer
(NK) cells; and hematologists interested in
the formation of myeloid cells, which encom-
pass monocytes, macrophages, and different
classes of neutrophils as well as red blood cells
and platelets. However, these camps have con-
verged to some extent in recent years, and the
need for such a merger is perhaps no more ev-
ident than when one tries to understand the
molecular basis of lineage diversification be-
tween lymphoid and myeloid cells. The estab-
lishment of all hematopoietic lineages during
development is mediated by transcription fac-
tors that act in sequential and parallel fashions,
building lineage-specific networks or circuits
(for reviews, see 1–4). Logically, the lymphoid
and myeloid networks intersect at the devel-
opmental branching points between these lin-
eages. This review focuses on transcription
factors and other parameters, such as cytokine
receptor signaling, that have an instructive
role at lymphoid-myeloid junctures. In the
following section, we give an overview of sev-
eral current models of hematopoietic lineage
trees to pinpoint developmental branching
points between the lymphoid and myeloid cell
The stochastic model of hematopoiesis states
that a single multipotent progenitor (MPP)
has the option to differentiate along more
than two pathways. This early model was
based on the observation that colony as-
says, using single myeloid progenitors, yielded
highly heterogeneous outcomes (5). As dis-
cussed below, most current models imply
that hematopoietic differentiation proceeds
along an ordered pathway with binary deci-
sion steps. However, ordered binary choices
are not apparent, at least during the earliest
stages of differentiation.
The Akashi-Kondo-Weissman
Scheme of Hematopoietic
The identification of stem and progeni-
tor cells by Weissman and collaborators
(6–9, 13, 14) led to the construction of a
hematopoietic lineage tree that is charac-
terized by a cascade of binary decisions
(Figure 1a). The staining of bone marrow,
with a combination of cell surface antigen-
specific antibodies led to the prospective
isolation of hematopoietic stem cells (HSCs)
as lin
(LSK) cells (6–8).
These cells can be further subdivided into
long-term HSCs (Thy-1
short-term repopulating HSCs (Thy-
), and MPPs (Thy-1
) (9),
populations that were also defined by other
combinations of markers (10–12). Similar
approaches led to the identification of
progenitors with a more restricted differen-
tiation potential. Thus, in the bone marrow
investigators identified a common progenitor
for all lymphoid lineages (CLP) (13), as well
as a common myeloid progenitor (CMP)
that generates granulocytic-macrophage
(GM) and megakaryocytic-erythroid (MegE)
lineages (14). CLPs give rise to pro-B and
pro-T cells, uncommitted lymphoid pro-
genitors that will differentiate further into
mature B and T cells (13). CLPs also
produce NK lineage cells (13). CMPs in
turn generate two more restricted progen-
itors: granulocyte-macrophage progenitors
(GMPs) and megakaryocyte-erythrocyte pro-
genitors (MEPs), generating GM and MegE
cells, respectively (14). The offspring of
GMPs also includes neutrophils, eosinophils,
and possibly basophils/mast cells (15, 16).
The observation that CMPs and CLPs
derived from adult bone marrow generate
mutually exclusive progeny (13, 14) sug-
gests that their diversification represents the
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B cell
T cell
NK cell
dendritic cells
Figure 1
Lineage trees of adult hematopoiesis and lymphoid-myeloid branching points. (a) The
Akashi-Kondo-Weissman model of adult hematopoiesis (13, 14) with the branching point between
lymphoid and myeloid lineages indicated by the gray shaded circle. (b) Revised lineage tree, showing
three areas where branching might occur (LT- and ST-HSC, long-term and short-term HSC; MPP,
multipotent progenitor; ETP, early T lineage progenitor).
Lymphoid-Myeloid Development 707
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ETP: early T
lineage progenitor
double negative;
DN1–DN4: stages
of DN cell
commitment: the
process by which cell
fates are determined
earliest branching point during hematopoietic
Adult Lympho-Myeloid Progenitors
Lacking Erythroid and
Megakaryocytic Potentials?
Although the Weissman scheme has proven
invaluable for the conceptual understand-
ing of hematopoiesis, more recent experi-
mental data suggest that alternative devel-
opmental pathways generating myeloid and
lymphoid cells exist (Figure 1b). The recent
description of macrophage–/T cell–/B cell–
restricted progenitors lacking MegE potential
by Jacobsen and colleagues (17) is particularly
intriguing. These cells resemble previously
described MPPs because they are contained
within the LSK population and express high
levels of Flt3. The model proposed by Ja-
cobsen and colleagues implies that GM cells
can be generated by two distinct pathways:
one via classical CMPs and one via lympho-
myelomonocytic progenitors without MegE
potential. An early branching between MegE
and lymphoid-myelomonocytic cells might
reflect the choices of an ancestral HSC be-
cause lymphocytes are evolutionarily younger
than GM and MegE cells (18). However, in
a reassessment of the differentiation poten-
tial of Flt3
MPPs, a transient MegE poten-
tial was detectable both in culture and after
transplantation (C.E. Forsberg, T. Serwold, S.
Kogan, I.L. Weissman & E. Passegue, per-
sonal communication).
Uncertainty About the Physiology
and the Lineage Restriction of T Cell
Unlike all other lineages, which are specified
in the bone marrow, T cells differentiate af-
ter migration of early progenitors into the
thymus. Bone marrow–derived CLPs can dif-
ferentiate into T lymphocytes in fetal thymic
organ culture (FTOC) and after intrathymic
injection (19). Another clonogenic B/T bipo-
tent progenitor termed CLP-2, which coex-
presses pre-T
(a T lineage marker) and B220
(a B lineage marker), was later identified in
bone marrow and shown to seed the thymus
after intravenous transfer (19a). However, the
physiological relevance of the CLP pathway
has recently been called into question by the
finding of Bhandoola and colleagues (20) that
the earliest T lineage progenitors (ETPs),
contained within the CD44
fraction of CD4/CD8 double-negative (DN)
thymocytes, have a more robust T cell recon-
stitution capacity than CLPs. The same study
showed that mice deficient for the Ikaros tran-
scription factor lack CLPs but not ETPs (20),
although it cannot be ruled out that in these
mice CLPs are present but lack the expression
of one of the markers that serve to identify
them. Although ETPs also generate B cells
with delayed kinetics, they are not lymphoid
restricted because they can generate myeloid
cells in vitro at low frequencies (20). Later,
the same authors also reported that CLPs are
absent from peripheral blood and that the
sole population with T lineage potential has
an HSC phenotype, again suggesting a CLP-
independent pathway for T cell differentia-
tion (21). Together, these results suggest that
T cells can develop by at least two distinct
pathways, CLPs and ETPs (22).
If HSCs are the thymus-seeding cells,
the question arises as to when they lose
their myeloid and B lymphoid potential. That
lineage commitment occurs at the pro-T
(DN1/DN2) to pre-T (DN3/DN4) transi-
tion is well established (23). Several reports
indicate that pro-T cells can form NK and
dendritic cells at the single-cell level (24, 25),
although their B cell potential is more con-
troversial. One report describes the existence
of bipotent T/B progenitors in the thymus
and confirmed their identity in single-cell as-
says (25). However, two other studies failed to
detect B cell potential in thymic progenitors
with robust T lineage potential (26, 27). Us-
ing limiting dilution assays, one of these stud-
ies reported the generation of Mac-1
myeloid cells from a thymic progenitor with-
out B cell potential (27), suggesting that a
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general lymphoid-myeloid branching point
does not exist during the generation of T cells,
and that B cell potential is lost before myeloid
potential. Thus, the thymic microenviron-
ment appears to suppress alternative fates in
an ordered fashion (28).
Multiple Origins of Dendritic Cells
Dendritic cells can be broadly classified as
“myeloid” dendritic cells and
“lymphoid” dendritic cells (29). Spe-
cialized subtypes of dendritic cells also exist,
including Langerhans dendritic cells found in
the skin and plasmacytoid dendritic cells (29).
Curiously, functionally equivalent and pheno-
typically indistinguishable myeloid and lym-
phoid dendritic cells can be derived from ei-
ther CMPs or CLPs (30, 31). In fact, progen-
itors downstream of CMPs and CLPs such as
GMPs and pro-T cells but not pro-B cells can
still give rise to these two classes of dendritic
cells (31). Although molecular differences be-
tween CMP- and CLP-derived dendritic cells
might exist, their dual origin suggests that
myeloid and lymphoid progenitors can con-
verge into the same phenotype and therefore
do not have mutually exclusive differentiation
Lineage Trees in the Fetal Liver:
Dissent and Consensus
To analyze the differentiation of single fe-
tal liver–derived LSK cells, Katsura and col-
leagues (32–34) used an in vitro culture sys-
tem consisting of a modified FTOC, which
measured macrophage (M), erythroid (E),
B cell, and T cell differentiation poten-
tials simultaneously. They found progenitors
with various potentials, including M/E/T/B,
M/E, M/T/B, M/T, and M/B, as well as
monopotent progenitors (32–34). However,
T/B progenitors were never found, even
when the fraction corresponding to bone mar-
row CLPs (lin
) was examined
(34). These observations challenged the no-
tions that CLPs exist in the fetal liver and
progenitor: a cell
capable of
differentiation into a
number of defined
lineages but without
Cytokines: growth
factors secreted by
cells that bind to
specific receptors,
induce cell signaling,
and result in survival,
growth, and
that hematopoietic commitment must entail
an early myeloid-lymphoid branching point
(Figure 2a). However, because researchers
could not define the phenotype of the indi-
vidual progenitor subtypes, these cells could
not be prospectively purified and tested in
other assays. Akashi and colleagues (35, 36)
also sought to identify intermediate progen-
itors in the fetal liver and found populations
with similar cell surface characteristics as bone
marrow CLPs and CMPs. In addition to the
expected progeny of these cells, limiting di-
lution analysis in vitro showed that 1 in 14
CLPs generated macrophages but not MegE
lineage cells (35). Fetal CMPs gave rise to B
cells, but not T cells, at a frequency of 1 in
160 cells (36) (Figure 2b). Because the for-
mer study did not simultaneously test the B,
T, and myeloid potentials of fetal CLPs at the
single-cell level, questions as to whether the
myeloid potential in this fraction segregates
with T or B potential and whether T/B pro-
genitors exist could not be addressed. An open
question therefore remains whether the fetal
liver M/T/B progenitors (34) are equivalent
to the low-fidelity CLPs (35). Nevertheless,
there is a consensus that the biological proper-
ties of the intermediate fetal progenitors differ
from those in the bone marrow, implying that
fetal and adult lymphoid-myeloid branching
points are not equivalent.
Lineage commitment could be induced either
by extracellular factors, including cytokines,
direct cell-cell interactions, or other environ-
mental cues. Alternatively, it could be induced
by intrinsic mechanisms, such as the stochastic
upregulation of transcription factors, or other
regulatory molecules, such as microRNAs (see
MicroRNAs: An Emerging Group of Putative
Lineage Determinants). Both extrinsic and
intrinsic factors may either have an instruc-
tive role and actively induce commitment and
differentiation or be merely permissive for
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B cell
T cell
B cell
T cell
NK cell
Figure 2
Lineage trees of
fetal hematopoiesis
branching points.
(a) Lineage tree
proposed by
Katsura (32),
showing two
separate and late
branching points
between lymphoid
and myeloid
lineages. (b) Model
based on the work
by Akashi and
(35, 36), in which
CMPs and CLPs
retain plasticity.
Branching points
are indicated by
gray shaded circles.
the outgrowth of precommitted progenitors
by promoting cell survival and/or expan-
sion (37, 38). In fact, evidence discussed
below suggests that both cytokine receptor
signaling and transcription factors have in-
structive roles in a cell context–dependent
At first glance, the phenotypes of mouse mod-
els deficient for different myeloid cytokines
and cytokine receptors suggest a permissive
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role for cytokine signaling, as these mice typ-
ically do not display a complete loss of the
associated lineage. For example, mice defi-
cient for the genes encoding G-CSF, GM-
CSF, or EPO display minor to significant re-
ductions in numbers of progenitors and ma-
ture progeny (39–41). At least in myeloid
differentiation, for which multiple receptors
have been identified, this could be attributed
in part to overlapping patterns of expression
of functionally redundant receptors. How-
ever, the phenotype of mice deficient for
both G-CSF and GM-CSF or M-CSF and
GM-CSF argues against this explanation be-
cause these mice did not develop a more
severe phenotype than G-CSF- or M-CSF-
deficient mice (42, 43). Observations gained
from studies in which the EPO receptor
(EpoR) and M-CSF/CSF-1 receptor (c-fms)
were overexpressed also support the permis-
sive model. Thus, expression of constitutively
active EpoR on MPPs derived from bone mar-
row did not skew differentiation toward the
erythroid lineage (44). Likewise, expression of
CSF-1R in MPPs was compatible with differ-
entiation into megakaryocytes and erythroid
cells (45). Additional experiments tested the
instructive capacity of a chimeric receptor
consisting of the extracellular domain of the
TPO receptor (TpoR or c-mpl) and the in-
tracellular signaling domain of the G-CSF
receptor (G-CSFR), expressed from the mpl
genetic locus. Expression of this molecule
rescued the thrombocytopenic phenotype of
mpl-deficient mice without promoting gran-
ulopoieisis (46). This result indicates that G-
CSFR can substitute for mpl signaling and
does not specifically instruct granulocyte dif-
ferentiation, but rather provides a generic sur-
vival function. Using a similar approach, the
signaling component of the EpoR in a chimera
with the ligand-binding domain of G-CSFR
did not redirect differentiation toward the
erythroid lineage nor disturb the formation of
granulocytes when activated by G-CSF (47).
Despite these results supporting the
permissive model, ectopic cytokine signaling
can act in an instructive fashion and is capable
MicroRNAs are small regulatory RNA molecules that bind
target sequences in messenger RNAs and inhibit their expres-
sion either by inducing their degradation or by inhibiting their
translation (182). The finding that microRNAs play a role in
differentiation and cell growth in plants and in invertebrates
(183) suggested similar functions in mammals. Indeed, miR-1
expression has been found to be activated during myocyte
differentiation by the transcription factors SRF, MyoD, and
MEF2, and this RNA in turn negatively regulates the tran-
scription factor Hand2. As a result, proliferation of cardio-
genic precursors is inhibited, reducing the number of ven-
tricular cardiomyocytes (184). About 100 microRNAs have
been found so far that are expressed in the hematopoietic sys-
tem, and their combinations are highly diagnostic for differ-
ent cell lineages and types of leukemia (185). miR-181 is most
abundant in B lineage cells. When multipotent hematopoietic
progenitors overexpressing the microRNA were transplanted
into irradiated mice, an increase of B cells relative to T cells
was observed (186). This finding raised the possibility that
miR-181 targets genes that are critical for the decision mak-
ing between lymphoid lineages. However, the effects observed
may reflect an increased survival/proliferation of a subset of
precommitted MPPs expressing the microRNA.
stimulating factor
stimulating factor
stimulating factor
of reprogramming restricted progenitors in
certain cellular contexts. One study described
the effects of expressing human CSF-1R
in murine pre-B cell lines. B cell factor–
independent sub-lines arose after prolonged
culture that spontaneously gave rise to
macrophage-like cells. These cells lost all
markers characteristic of the B cell lineage and
could be expanded in human (but not mouse)
M-CSF (48). More recent studies using
primary progenitors derived from transgenic
mice have extended the instructive role of
cytokine signaling to nonimmortalized pro-
genitors (Figure 3). In an attempt to induce
NK cell differentiation in early lymphoid pro-
genitors, Kondo et al. (19) analyzed the dif-
ferentiation potential of bone marrow CLPs
derived from transgenic mice expressing the
chain of the human IL-2 receptor (IL-2R
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GM cell
dendritic cell
CLP or
CLP or
Figure 3
Reprogramming of early lymphoid cells by instructive cytokine signaling.
Scheme illustrating how exogenous expression of human IL-2R followed
by ligand stimulation converts CLPs and pro-T cells into
myelomonocytic cells (top). IL-2R signaling leads to upregulation of
endogenous GM-CSFR
, which likely contributes to myelomonocytic
differentiation in this context (13, 50). As illustrated at the bottom, ectopic
GM-CSFR expression and stimulation is sufficient for this conversion and
also converts T cell progenitors into myeloid dendritic cells (49).
Surprisingly, a significant proportion of
-expressing CLPs formed GM
colonies after culture in medium containing
human IL-2, whereas wild-type CLPs formed
exclusively B cell colonies. IL-2R signaling
did not serve solely as a survival signal for
myelomonocytic progenitors because expres-
sion of Bcl-2 in CLPs did not induce the
formation of GM colonies. The effects of IL-
2R signaling might be mediated in part by the
induction of the GM-CSFR
gene because
it was upregulated in stimulated IL-2R trans-
genic CLPs (19). Ectopic expression of both
components of the human GM-CSFR (
c chains) in CLPs, followed by human
GM-CSF stimulation, induced a similar CLP
to GM conversion, as well as the formation
of myeloid dendritic cells (19, 49) (Figure 3).
Myeloid-instructive effects of IL-2R and
GM-CSFR signaling could also be observed
with pro-T cells, but not with pre-T cells or
pro-B cells (49, 50). Strikingly, the instructive
effect of GM-CSFR signaling is cell context
dependent, as transgenic MEPs expressing
hGM-CSFR were undisturbed by hGM-CSF,
indicating that GM-CSFR signaling is per-
missive for commitment of the MegE lineages
(49). Moreover, GM-CSF signaling could not
rescue defects in B and T cell differentiation
caused by disruption of lymphoid cytokine
IL-7 signaling (49), indicating that it is not
a generic survival signal. Together, these
experiments show that the IL-2 and GM-
CSF receptors can have lineage-instructive
potential and suggest that early lymphoid
and pro-T cell progenitors exhibit a latent
myeloid developmental capacity. They also
indicate that GM-CSFR, which is known
to be expressed on a subset of HSCs, must
be downregulated to permit commitment of
MPPs into lymphoid precursors.
Although multiple myeloid cytokine re-
ceptors have been identified, a smaller reper-
toire of receptors has been described for
lymphoid cells. Flt3 is first expressed on two
subsets of LSK cells, short-term HSCs and
MPPs that have lost the ability to self-renew
(51). Flt3-expressing MPPs rapidly differen-
tiate into B and T lineage cells, and mice defi-
cient in Flt3 or its ligand Flt3L have reduced
numbers of B and T progenitors (51–53). Im-
portantly, CLPs, but not HSCs or CMPs, are
severely decreased in these animals, suggest-
ing that Flt3 signaling fosters formation of
downstream lymphoid progenitors (53). The
chain is expressed on CLPs as well
as early B cells and T cell progenitors, and
IL-7 stimulates their proliferation. Analysis
of mice deficient in IL-7 or one subunit of
its receptor (IL-7R
) revealed severe defects
in B and T lymphoid differentiation, implicat-
ing IL-7 as a nonredundant cytokine (54, 55).
IL-7 functions, at least in part, by inducing ex-
pression of Bcl-2, thereby promoting survival
of lymphoid progenitors (56, 57). However,
IL-7 also influences differentiation of CLPs
because CLPs from IL-7-deficient mice dif-
ferentiate into T and NK cells but not B cells
(58). Interestingly, mice lacking both IL-7R
and Flt3 show a total absence of B cells in both
fetal and adult development (59), raising the
possibility that they act in an instructive fash-
ion, perhaps by activating B cell transcription
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factors (2). In support of this hypothesis, en-
forced expression of early B cell factor (EBF)
in CLPs derived from IL-7-deficient mice re-
stores B cell differentiation potential (58), and
IL-7 stimulation of pre-pro-B cells derived
from these mice rapidly induces expression of
EBF (58a).
Whether lineage decisions are induced by
extracellular cues, by intrinsic events, or by
a combination of both, they always involve
changes in gene expression programs. Because
these programs are ultimately controlled by
transcription factors, new cell fates must be
induced by changes in the expression or ac-
tivity of these factors. Before discussing ex-
periments showing how transcription factors
both activate novel gene expression programs
and extinguish existing ones in a lineage-
instructive fashion, we give a brief overview
about transcriptional regulators with known
or suspected roles in lineage commitment
Transcriptional Regulators Required
for Myeloid and Lymphoid
Figure 4 compiles a list of regulators whose
ablation affects the formation of intermedi-
ate hematopoietic progenitors or inhibits dif-
ferentiation or function of mature cells from
diverse hematopoietic lineages. The figure
does not include factors that act primarily on
HSCs. Five types of phenotypes, observed in
either conventional or conditional knockout
studies, are described: (a) complete loss of a
progenitor or lineage, (b) maturational or dif-
ferentiation block, (c) functional defect, and
(d ) decreased or (e) increased numbers of cells
in a given lineage. The reader is also referred
to more detailed reviews on transcription fac-
tors in the B cell lineage (2, 60, 61), T cell
lineage (23, 62, 63), and myeloid lineages (4,
Early Lymphoid and Myeloid
Ikaros and PU.1 are broadly expressed in the
hematopoietic system, including HSCs, early
lymphoid progenitors such as CLPs, and var-
ious myeloid lineages. Loss of either factor
disrupts B cell differentiation from uncom-
mitted progenitors (65–68). Ikaros regu-
lates transcription by recruiting co-repressors
and chromatin remodeling proteins to tar-
get genes (69). Although Ikaros deficiency
most severely affects the lymphoid lineages,
alterations can also be observed in myeloid
lineages (66, 69, 70). Ikaros may promote
lymphoid cell fates by activation of Flt3 ex-
pression and repression of GM-CSFR
pression in HSCs (71). How Ikaros acts in a
lineage-specific fashion is not well understood
but might be explained by the expression of
different splice isoforms in different lineages
and cooperation with the lymphoid-restricted
family member Aiolos (72–74). Loss of PU.1
causes a profound inhibition of B lineage and
myelomonocytic cell formation as well as de-
fects in T cell and dendritic cell differentia-
tion (67, 68, 75–77). Some of the broad ef-
fects of PU.1 deletion can be attributed to a
loss of CMPs and CLPs in these mice (78,
79). In addition, although HSCs are formed
in PU.1-deficient mice, and MegE cells are
still produced, PU.1 deletion in conditional
knockouts reduces the competitive repopula-
tion potential of HSCs (78). C/EBP
, whose
lineage-associated phenotype is discussed be-
low, has the opposite effect in that its loss en-
hances their repopulation potential (80).
B Cell Lineage
B cell development requires a complex set
of transcription factors, namely PU.1, Ikaros,
EBF, E2A, and Pax5, and inactivation of any
of these factors yields a severe phenotype. The
dramatic impairment of B cell development in
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PU.1-deficient mice has been ascribed to lack
of expression of the PU.1 target genes IL-7R
and EBF (81, 82) because B cell differentia-
tion is impaired in mice deficient for either
of these genes (83, 84). However, PU.1 is not
strictly required for B cell formation because
culture of PU.1-deficient fetal liver cells led
to an outgrowth of IL-7-dependent B lineage
cells that express both IL-7R
and EBF and
that can mature into IgM
cells (84a). This
outgrowth was delayed compared with that
of wild-type cells, suggesting that PU.1 facil-
itates B cell lineage specification in conjunc-
tion with other factors such as Ikaros (2). Mice
lack decr matur
lack lack
func lack
func decr
65, 66, 70, 71, 138
67, 68, 75, 76,
77, 78, 115
93, 95, 164, 165
84, 85, 111
110, 111
99, 100, 101, 102
118, 119
80, 122
124, 125
126, 127, 128
130, 131, 132
116, 117, 137
133, 134, 135, 136
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in which PU.1 is conditionally inactivated in
the bone marrow lack CLPs despite normal
expression of IL-7R
on lin
cells (78).
Loss of EBF blocks B cell development
at the pro-B cell stage before initiation of
B cell receptor (BCR) rearrangements (83).
A similar phenotype was described for E2A-
deficient mice (85) and for transgenic mice
overexpressing Id1, an inhibitor of E2A and
other helix-loop-helix (HLH) proteins (86).
E2A forms homodimers in B cells and collab-
orates with EBF at target gene promoters (87,
88). It probably also plays a role in growth
control because expression of the HLH in-
hibitor Id3 in pro-B cells limits their pro-
liferation in vitro (89). Target genes of E2A
encode proteins necessary for BCR rearrange-
ment, assembly, and signaling (63), a fact that
explains the observed block of differentiation
at the pro-B cell stage. EBF appears to be up-
stream of Pax5 because it binds to the Pax5
promoter and activates its expression in re-
porter assays (90, 91). However, the regula-
tory network of B cells clearly involves both
the sequential and concerted action of tran-
scription factors. This is exemplified by the
mb-1 gene, which is regulated by E2A and
EBF, as well as by Pax5 in cooperation with
Ets-1 (92).
mice display a block of B cell dif-
ferentiation at the pro-B cell stage, just after
initiation of BCR heavy chain rearrangements
(93). Pax5
pro-B cells express both EBF
and E2A (94), demonstrating that neither of
these factors nor their combination is suffi-
cient for B cell maturation. Most interestingly,
these cells also express myeloid genes, such as
, as well
as the T cell transcription factor Notch1, in-
dicating that an important role of Pax5 is to
repress lineage-inappropriate gene expression
(95–97). E2A
pro-B cells also inappropri-
ately express myeloid genes. The relevance of
these findings with regard to lineage commit-
ment is discussed below.
T Cell Lineage
In mice lacking either GATA-3 or Notch1,
T cell development is arrested at the earliest
discernable T cell progenitor stage (98, 99).
Chimeras between GATA-3-deficient embry-
onic stem cells and wild-type blastocysts
showed contributions of knockout cells to
all hematopoietic lineages except T cells (99,
100). Besides a role in T cell specification,
conditional knockouts revealed functions of
GATA-3 at various stages of T cell matura-
tion (101, 102). Notch1 is a transmembrane
receptor that, after ligand binding and cleav-
age, translocates to the nucleus and regu-
lates transcription by converting its nuclear
cofactor CSL into a transcriptional activa-
tor. Notch1 inactivation or that of CSL leads
Figure 4
Hematopoietic cell phenotypes of mice lacking transcriptional regulators. The figure lists transcription
factors with proven or suspected roles in lineage commitment. Shaded boxes indicate altered phenotypes
of the cell types indicated above, as observed after conventional (germ line) and conditional (Mx1-Cre or
lineage-specific-Cre) gene inactivation of each regulator. Lack of shading indicates that no phenotype
was observed or, in some cases, that defects were not studied. The altered phenotypes were either a
complete loss of a lineage (lack), a maturational block (matur), a functional defect ( func), decreased
numbers of lineage cells (decr), or increased numbers of lineage cells (incr). For PU.1, results from
conventional and conditional knockout differ as to whether the factor is required in B cell development.
For both Ikaros and PU.1, null phenotypes completely lack fetal but not adult T cells. FOG-1-deficient
embryos lack megakaryocytes and display a maturational defect in erythroid cells. GATA-1-deficient
embryos lack erythroid cells and display a maturational defect in megakaryocytes. Relevant references are
listed in the right column. Abbreviations: ZnF, zinc finger domain; HTH, helix-turn-helix domain;
HLH, helix-loop-helix domain; transmem, transmembrane; HMG box, high motility group box; bZip,
basic leucine zipper; RHD, Rel homology domain. Lineage abbreviations are as used throughout the
text, with DC indicating dendritic cells.
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to an early block in T cell development
(98, 103). Conversely, expression of constitu-
tively active Notch1 in bone marrow chimeras
induces T cell differentiation in the bone
marrow at the expense of B cell differentia-
tion (104). Whether Notch1 signaling specif-
ically instructs T cell lineage commitment
or rather promotes survival/proliferation of
precommitted progenitors remains a mat-
ter of contention. Expression of activated
Notch1 in fetal liver–derived hematopoietic
progenitors or their culture on OP9 stro-
mal cells that constitutively express Notch
ligand delta-like-1 (DL1) induces T cell
differentiation and inhibits differentiation
into alternative lineages such as B and
NK cells (105, 106). High Notch signaling
strengths are required to exclude differen-
tiation into NK cells, whereas weak Notch
signals are sufficient to inhibit B cell differen-
tiation (106). Nevertheless, when hematopoi-
etic progenitor cells are cultured in T permis-
sive culture conditions and then switched to
B permissive conditions, some cells still retain
B potential, indicating that Notch is not suffi-
cient to drive irreversible commitment (107).
E2A forms heterodimers with HEB at all
stages of thymocyte development. The com-
plex activates the transcription of genes asso-
ciated with T cell receptor (TCR) rearrange-
ment and signaling (108). In the absence of
either E2A or HEB, T cell differentiation is
partially blocked (109, 110). One HLH fac-
tor likely compensates for the other, as mice
expressing a dominant-negative form of HEB
(which sequesters E2A or HEB into inactive
heterodimers) show a more severe T cell phe-
notype than mice that lack either factor alone
(111). Moreover, a role for E2A in commit-
ment is inferred from experiments in which
expression of Id3 in progenitors diverts T cell
differentiation into the NK lineage (112).
Although GATA-3, Notch1, and E2A/
HEB are all expressed in pro-T cells, T cell
commitment does not occur until the pre-
T cell stage, when all other developmental
options are exhausted (23). This fact suggests
that T cell commitment requires an additional
event, perhaps involving the downregulation
of PU.1, which occurs at the transition be-
tween pro-T and pre-T cells (23, 113, 114).
The observation that overexpression of PU.1
in hematopoietic progenitors limits their ex-
pansion in FTOC and promotes myeloid
differentiation at the expense of T cell dif-
ferentiation supports this suggestion (114).
However, additional positive or negative reg-
ulators may exist that play a role in this
Natural Killer Cell Lineage
Ikaros-, PU.1-, and Id2-deficient mice all pos-
sess severe NK lineage defects. In Ikaros-
deficient mice, T lymphocytes but not NK
cells are produced in adult animals, sug-
gesting an NK-specific defect (66). PU.1-
deficient mice display a more subtle pheno-
type, with reduced numbers of NK cells that
also have functional defects (115). In Id2-
deficient mice, NK cells in the periphery are
severely reduced (116, 117). An instructive
role of Id proteins in NK cell development
is also implied by experiments in which Id3
expression in human CD34
progenitors promoted NK cell development
at the expense of T cell development (112).
In this case, Id3 likely mimics the function of
Id2 in the physiological context because mice
lacking Id3 do not exhibit a NK-specific phe-
notype (C. Murre, personal communication).
Id3-deficient mice also display defects in T
and B cell maturation (118, 119).
Granulocytic-Macrophage Lineages
Many macrophage- and granulocyte-res-
tricted promoters are regulated by PU.1
and/or C/EBP
(120). These factors coop-
erate in the regulation of the genes encod-
ing the myeloid growth factor receptors M-
CSFR, G-CSFR, and GM-CSFR (64, 120).
In PU.1-deficient mice, all myelomonocytic
cells are absent. However, as in the B cell lin-
eage, PU.1 is not strictly required for commit-
ment because immature myeloid precursor
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lines dependent on IL-3 can be established
from PU.1-deficient fetal liver (121). Condi-
tional deletion of PU.1 in adult bone marrow,
using the Mx1-Cre deleter system, leads to
a complete loss of CMPs and GMPs (78, 79).
Mice deficient in C/EBP
lack neutrophil and
eosinophil granulocytes, and conditional in-
activation in the bone marrow shows the spe-
cific absence of GMPs and reduced numbers
of CMPs, leading to decreased formation of
all downstream lineages (80, 122). In colony
assays, the remaining CMPs yielded normal
MegE colonies and GM colonies containing
cells with an immature phenotype (80). Dele-
tion of C/EBP
by retroviral expression of
Cre recombinase in GMPs does not prevent
their terminal differentiation (80), showing
that this factor is not needed at late stages, per-
haps because its function is replaced by that
of other C/EBP family members. The func-
tions of C/EBP
in granulocyte formation ap-
pear to be redundant with its close relative
because C/EBP
expressed from
the C/EBP
locus rescues neutrophil granu-
locyte development (123). Macrophages from
-deficient mice develop normally but
display defective bacterial cell killing (124).
Unlike C/EBP
is also expressed
in B cells, and C/EBP
-deficient mice display
defects in B cell expansion (125).
Megakaryocytic and Erythroid
In GATA-1-deficient embryos, development
of erythroid cells is blocked early in differ-
entiation, leading to a lethal anemia (126,
127). Expression of both GATA-2 and GATA-
3 transgenes rescued the erythroid lineage
defect in GATA-1-deficient mice but also
revealed nonredundant roles of GATA-1 in
erythroid maturation (128). GATA-1 is also
required for the maturation of the megakary-
ocytic lineage, a finding that was only made
after the gene was specifically inactivated in
megakaryocytes (129). GATA-1 collaborates
with FOG-1 (friend of GATA-1) during ery-
factor network:
repertoire of
transcription factors
that interact both
positively and
negatively to
establish and
maintain gene
expression programs
thropoiesis, and loss of FOG-1 leads to a par-
tial block in erythroid differentiation and a
complete loss of megakaryocytes (130, 131).
The more severe megakaryocyte phenotype
of FOG-1-deficient compared with GATA-
1-deficient mice suggests that FOG-1 has
GATA-1-independent functions in this lin-
eage. FOG-1 expression is not completely re-
stricted to megakaryocytes and erythroid cells
but is also present in late stages of T cell devel-
opment, when it negatively regulates GATA-3
Dendritic Cell Lineages
In a number of transcription factor knock-
out mice, dendritic subtypes are decreased
in number or completely absent. Besides mi-
nor defects in B cell maturation (133), RelB
knockouts specifically lack Mac-1/CD11b
dendritic cells (134–136). In contrast, Id2-
deficient mice lack CD8
dendritic cells as
well as Langerhans cells, and CD11b
dritic cells are also reduced in these mice
(137). Mice carrying a dominant-negative
form of Ikaros lack both types of den-
dritic cells (138). Finally, PU.1-deficient mice
lack Mac-1/CD11b
dendritic cells, although
there are conflicting reports as to whether
dendritic cells are also affected (76,
As is evident from the above section, some
transcription factors are required in multiple
cell types, and more than one transcription
factor is required to specify a given lineage.
These requirements suggest that each lin-
eage is defined by a specific transcription
factor combination and, as discussed below,
by specific regulatory interactions, compris-
ing the transcription factor network. The ul-
timate proof for the instructive capacity of
a transcription factor is the demonstration
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Transcription factors activate genes not only by recruiting
coactivators to the basic transcriptional machinery, but also
by altering chromatin through the formation of complexes
with enzymes that modify and remodel chromatin (187). In
addition, they recruit co-repressors and chromatin-modifying
factors when they repress genes. In particular, the activating
and repressive functions of a number of hematopoietic fac-
tors, including Ikaros, PU.1, Runx1/AML1, C/EBP
, Pax5,
and GATA-3, have been linked to changes in chromatin ar-
chitecture (69, 141d, 188–192). Specifically, at the IgH locus,
Pax5 alters histone methylation (188), whereas PU.1 mod-
ulates chromatin accessibility of IgH enhancers (189). The
actions of these transcription factors at specific loci are devel-
opmentally regulated. A well-studied example is the M-CSFR
(c-fms) locus. This gene is expressed in multilineage progen-
itors, where it is in an active state accessible to nucleases
and bound by transcription factors (which include PU.1 and
C/EBP). However, in cells where M-CSFR becomes downreg-
ulated, such as in mature B cells, the enhancers no longer bind
the critical transcription factors, and the locus becomes nucle-
ase insensitive (193). The ability of instructive transcription
factors to reprogram cells and effectively make over estab-
lished gene expression programs likely is limited by the ac-
cessibility of promoter/enhancer elements embedded within
that it can reprogram a committed cell into
another lineage by perturbing its transcription
factor network, deconstructing the old one
and reconstructing it into a new one. In this
logic, transcription factor network perturba-
tions in committed cells to probe mechanisms
of cell programming can be compared to in-
ducing mutations to probe a gene’s function.
Similar experiments, performed with MPPs,
cannot unambiguously demonstrate instruc-
tive effects of a molecule because cell se-
lection, rather than instruction, is always a
possibility. In the following sections, we dis-
cuss mechanisms that enable a single tran-
scription factor to change a cell’s transcription
factor network.
Myeloid Cell Reprogramming: The
GATA-1:PU.1 Paradigm
Several principles of how transcription fac-
tors control hematopoietic cell fates have
been derived from studies of avian progeni-
tors transformed with the E26 virus, which
expresses the Myb-Ets oncogene. These cells,
which differentiate into either erythrocytes
or thrombocytes (avian megakaryocyte equiv-
alents) when the oncoprotein is inactivated,
are termed MEP
cells and can be induced
to differentiate into myeloblasts (macrophage
precursors) by activation of the Ras or pro-
tein kinase C pathways (139). Concomi-
tant with this transition into myeloblasts,
the cells downregulate the MegE regulator
GATA-1. Enforced expression of GATA-1 in
myeloblasts induces the formation of MEP
cells (140). Conversely, enforced expression of
PU.1, a transcription factor whose high-level
expression regulates myelomonocytic genes,
reprograms MEP
cells into myeloblasts
(141) (Figure 5). Thus, simply changing the
balance of two lineage hematopoietic tran-
scription factors can lead to the reversible re-
programming of committed myeloid cells. A
simple scheme that explains the role of tran-
scription factor stoichiometry in hematopoi-
etic lineage decisions is depicted in Figure 6a
and 6b. GATA-1 and PU.1 extinguish the
alternative cell fate by antagonizing each
other via protein-protein interactions (141a–
c). The inhibition of GATA-1 by PU.1 en-
tails recruitment of the Rb protein to the
GATA-1:PU.1 complex bound to erythroid
regulatory sequences, followed by chromatin
remodeling and the formation of a represso-
some (141d). Other interactions of transcrip-
tion factors with chromatin are discussed in
The Transcription Factor–Chromatin Con-
nection. GATA-1 antagonizes PU.1 by block-
ing the interaction between PU.1 and its
cofactor c-Jun (142). A two- to three-day long
exposure of the active form of GATA-1 (or of
PU.1) is sufficient to induce the irreversible
commitment of myeloblasts into MEP
cells (or of MEP
cells into myeloblasts)
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(140, 141). This time frame suggests that
overexpressed factors activate their respective
endogenous counterparts, establish an au-
toregulatory loop, and also interrupt autoreg-
ulation of the antagonistic factor. A diagram
that summarizes the GATA-1:PU.1 antago-
nism is shown in Figure 6c. Although there
is strong evidence for PU.1 autoregulation
(143), the situation for GATA-1 is less clear
because deletion of a high-affinity GATA-1-
binding site in the GATA-1 promoter mostly
affects eosinophil development, but it only
weakly affects erythroid cell numbers (144).
Similar changes in cell fates can also be
induced in primary, nontransformed cells.
For example, enforced expression of GATA-
1 in GMPs induced the formation of MegE
colonies in a high percentage of the cells
(145). In addition, expression of GATA-1 in
density-selected c-kit
cells that have
restricted GM potential induced the forma-
tion of MegE colonies and also eosinophil
and basophil colonies (146). Importantly, in-
vestigators have recently shown with mor-
pholino technology in zebrafish that lowering
the dosage of GATA-1 (but not of GATA-2)
induces a conversion of erythroid cells into
myeloid cells (147) and that reducing PU.1
levels induces a switch of myeloid into ery-
throid cells (148). These experiments also sug-
gest that the affected progenitors express both
transcription factors, whose balance is altered
by the antisense experiments. In conclusion,
myeloid lineage switching can be induced in
predictable ways not only by transcription fac-
tor overexpression, but also by their knock-
down, in both cases altering the balance be-
tween two antagonistic transcription factors.
Simple Transcription Factor
Combinations Establish Myeloid
Lineage Fates
The deceptively simple schemes in
Figures 6a, b, and c do not account for
the fact that a myeloid fate is determined by
combinations of cooperating transcription
factors in all lineages studied so far. This
PU.1 (C/EBP)
FOG-1 PU.1
Figure 5
Reversible myeloid lineage reprogramming in the avian MEP
The scheme represents experiments performed with cultured avian cells
transformed by the Myb-Ets oncoprotein (3). The arrows denote direction
of induced differentiation. Transcription combinations that are minimally
required for the specification of each cell type are indicated. MEP
and eosinophils show approximately a twofold difference in the level of
GATA-1 expression. Correspondingly, higher concentrations of GATA-1
are required to induce MEP
cell formation than to induce eosinophils
(140). PU.1 induces an eosinophil intermediate when temporarily expressed
in MEP
cells, as indicated by a dashed arrow (141).
was largely deduced from enforced tran-
scription factor experiments. For example,
in the avian system, expression of either
cells or of low amounts of
GATA-1 in myeloblasts induced eosinophil
formation. Conversely, expression of FOG-1
in eosinophils converted them into MEP
cells (for reviews, see 3, 149). Therefore,
the MegE lineage is specified by high levels
of GATA-1 in combination with FOG-1,
myelomonocytic cells by high levels of PU.1
together with C/EBP, and eosinophils by
moderate levels of GATA-1 and C/EBP
(Figure 5). A similar binary code was de-
scribed for the specification of mast cells:
Here, GATA-2 cooperates with PU.1 (150).
These cooperative interactions appear to be
reinforced by feed-forward mechanisms, at
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MegE progenitors
MegE genes GM genes
GM progenitors
Figure 6
Principles of transcription factor–mediated lineage commitment. (a) and (b) Role of stoichiometry in
MegE and GM lineage commitment as illustrated for the GATA-1:PU.1 paradigm. GATA-1 in excess
specifies MegE cell fate, and PU.1 in excess specifies GM cell fate. The illustration in (c) shows cross
antagonisms and autoregulatory loops of GATA-1 and PU.1, which lead to stabilization of cell
phenotypes during commitment. (d) Feed-forward mechanisms whereby GATA-1 activates expression of
its cooperating partner FOG-1 (145), and C/EBP activates PU.1 expression (120, 154), shown with solid
arrows. The possibility that C/EBP is activated by PU.1 is indicated by the dashed arrow.
least in specific cell contexts. For example, en-
forced GATA-1 expression in myeloblasts and
in GATA-1-defective erythroid precursors
upregulates FOG-1 expression (151, 152).
Although never directly addressed, PU.1 may
be able to induce C/EBP expression. Some of
these mechanisms may work in the opposite
direction because C/EBP expression upreg-
ulates PU.1 expression in myeloid cell lines
as well as in primary B cell precursors (153,
154). Feed-forward mechanisms (illustrated
in Figure 6d ) serve to stabilize lineage deci-
sions and to induce high-level expression of
lineage-restricted proteins such as beta globin
expression in erythroid progenitors [through
the cooperation between GATA-1 and
FOG-1 (152)] and of Mac-1 in macrophages
[through the cooperation between PU.1
and C/EBP (154)]. The cooperation be-
tween GATA-1 and FOG-1 strictly requires
specific protein-protein interactions (155).
Promoter/enhancer studies generally support
the concept that the pairwise combinations
of lineage-instructive transcription factors
described here, together with other more
broadly expressed transcription factors such
as Ets1, Runx1 (AML-1), and Myb, cooperate
in the regulation of lineage-restricted genes
Lymphoid-to-Myeloid Cell
Approaches similar to those used to study
the instructive effects of transcription
factors in myeloid lineages have recently led
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Day 0
Day 2
Day 4
Figure 7
-induced reprogramming of B cell progenitors over time as visualized by FACS analysis.
CD19-expressing B cell progenitors were isolated from the bone marrow, infected with a retrovirus
containing C/EBP
, and seeded under culture conditions permissive for both B and myeloid cell
development. As indicated by arrows, at day 2 after infection some cells downregulated CD19, others
upregulated Mac-1, and many did both. At day four about 60% of the cells converged on a CD19
population (arrows), also revealing a population of nonresponders. [Figure adapted from Xie
et al. (154).]
to the successful reprogramming of lymphoid
progenitors and to the elucidation of some
of the underlying mechanisms. Enforced
expression of GATA-1 in CLPs switched
40% of cells seeded singly in colony assays
into MegE colonies, and a similar conversion
was observed after transplantation into
irradiated hosts (145). In contrast, when
C/EBP was expressed in CLPs, they gen-
erated GM colonies (M. Kondo, personal
communication). Developmental plasticity in
the lymphoid compartment is not restricted
to the earliest lymphoid progenitors. A
link between B cell and granulocyte de-
velopment has been suggested by a mouse
line (Max41) in which the integration of a
transgene dramatically increased the number
of neutrophils at the expense of B cells. The
granulocytes of the transgenic line exhibit
immunoglobulin rearrangements, indicating
that they originated by a trans-differentiation
of B cells in vivo (160). How integration
of the transgene induces the switch is not
clear. Experiments in which the Raf or Ras
oncogenes were expressed in pro- or pre-B
cell lines showed that the cells switch at low
frequencies into functional macrophages
(161), suggesting that in these cells a lineage-
instructive myeloid transcription factor
had become activated. Reprogramming
into inflammatory-type macrophages could
indeed be induced by enforced expression
in B cell precursors by both C/EBP
in 60% of the cells, whereas PU.1
and other C/EBP factors showed little effect
(154). FACS analyses permitted the visualiza-
tion of the reprogramming process over time,
showing that reprogramming was initiated in
a stochastic manner, with individual cells up-
regulating Mac-1 or downregulating CD19
or both. After four to five days, all repro-
grammed cells had acquired a Mac-1
phenotype (Figure 7). The switch could also
be induced in immunoglobulin-expressing B
cells from the spleen, although at somewhat
lower frequencies. C/EBP requires PU.1 for
expression of at least some myelomonocytic
genes because PU.1
B lineage cells did
not upregulate Mac-1. In a feed-forward
mechanism, enforced expression of C/EBP
induced the upregulation of endogenous
PU.1 at levels comparable to those seen
in macrophages. The extinction of CD19
expression was PU.1 independent and was
due to the ability of C/EBP
to inhibit the
activity of Pax5 on the CD19 promoter (154),
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imm B
CD19 Mac-1
Figure 8
Mechanisms of transcription factor–induced B cell reprogramming. The
scheme illustrates that different stages of B cell differentiation, including
immature B cells (imm B), which express immunoglobulin, can be
reprogrammed into macrophages. The insert shows that C/EBP
extinguishes CD19 expression by inhibiting Pax5 activity (red T bar) and
induces myeloid gene expression by cooperating with endogenous PU.1
(green arrow), as well as by upregulating PU.1 expression.
probably owing to antagonistic interactions
between the two proteins (W. Ci & T.
Graf, unpublished observations). The results
described are illustrated in Figure 8. The
inhibition of Pax5 activity does not explain
the ability of C/EBP to induce a com-
plete collapse of the B cell gene expression
program. How this is achieved is not yet
entirely clear but may involve a secondary
antagonism by upregulated PU.1 (H. Xie
& T. Graf, unpublished results). In another
study, enforced expression of C/EBP
not cause myeloid reprogramming of B
cell precursors, although it did reprogram
B cell precursors (161a). The reason
for this apparent discrepancy to the previous
study is not obvious because, in an exchange
of vectors encoding C/EBP
, the construct of
Heavey et al. (161a) reprogrammed wild-type
B cell precursors at similar efficiencies as the
one reported by Xie & Graf (H. Xie & T.
Graf, unpublished observations). Commit-
ted T cells can also be reprogrammed by
into macrophages after enforced
expression, using an approach similar to
that used for the B lineage (C.V. Laiosa,
H. Xie & T. Graf, manuscript submitted).
Furthermore, T cell commitment is also
altered by overexpression of PU.1 or SpiB, a
transcription factor related to PU.1, either of
which converts pre-T (DN3) cells into cells
resembling dendritic cells (162, 162a).
The findings that CLPs and committed B
and T cell precursors can be switched into
myeloid cells beg the questions whether this
process is reversible and whether B and T cells
can be converted into one another. This was
studied using a knockin mouse line in which
Pax5 was expressed under the control of the
Ikaros locus, which is expressed in all lym-
phoid and myeloid progenitors (96). Pax5 did
not significantly disrupt myeloid or erythroid
differentiation, suggesting that in this con-
text it is insufficient for B cell specification.
However, T cell differentiation was disturbed,
with decreased thymic cellularity and a dif-
ferentiation block around the time of T cell
commitment. In reconstitution experiments
with lethally irradiated mice, bone marrow
progenitors expressing Pax5 did not con-
tribute to T cell differentiation. This might
be explained by the ability of Pax5 to in-
hibit Notch1 expression, as shown indirectly
by reintroduction of Pax5 into Pax5
cell precursors (96). Moreover, although Pax5
failed to interfere with the expression of the
T cell markers Thy-1 and CD3
in thymo-
cytes, it upregulated the B cell–specific gene
CD19 (96). In an independent study in which
Pax5 expression in T cells was driven by ele-
ments from the human CD2 promoter, Pax5
induced expression of its known targets as
well as heavy chain rearrangements without
initiating other aspects of the B cell pro-
gram (162b). Finally, experiments in which
primary macrophage cultures were infected
with retroviruses carrying either Pax5, E2A,
or EBF and cultured under conditions per-
missive for B cell development likewise did
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not induce the formation of nonadherent
CD19-expressing lymphoid cells (H. Xie & T.
Graf, unpublished observations). In sum, ec-
topic Pax5 expression in myeloid cells has no
detectable effects on their phenotype, whereas
it induces a differentiation defect in early
T cells and activation of some B cell–specific
target genes.
The effect of T cell transcription factors
on the phenotype of B cell precursors has not
been directly studied. However, seeding fe-
tal liver LSK cells onto OP9-DL1 stroma,
conditions under which Notch1 is activated,
inhibited their differentiation into the B cell
lineage, normally seen on OP9 cells not ex-
pressing DL1. Once cells were committed
to the B cell lineage, they were no longer
impeded by OP9-DL1 stroma (105). This
finding is consistent with the notion that
Notch signaling interferes with B cell com-
mitment but not with B cell proliferation and
Lymphoid Transcription Factors
Repress Lineage Inappropriate Gene
A serendipitous observation led to the dis-
covery that the inactivation of a lineage
commitment factor can awaken a dormant
multilineage potential in cells that were os-
tensibly specified to the B cell lineage. Thus,
when Pax5
pro-B cells were cultured for
several weeks on ST2 stromal cells (which
produce myeloid cytokines) without replen-
ishing IL-7, the formation of macrophages
was observed. The Pax5
pro-B cells also
had the capacity to differentiate into osteo-
clasts, granulocytes, NK cells, and dendritic
cells (95) under appropriate culture condi-
tions and into T cells upon transfer into im-
munodeficient recipients (95, 163). The mul-
tilineage potential (summarized in Figure 9)
was not brought about by selection in cul-
ture because freshly isolated Pax5
cells also generated T cell progeny after
transplantation (163). The B cell origin of
T cell
NK cell
dendritic cell
Figure 9
Multilineage differentiation potential of Pax5
and E2A
B cell
progenitors. See text for details.
these cells was supported by the detection of
immunoglobulin heavy chain DJ rearrange-
ments, although a small percentage of nor-
mal thymocytes also possess heavy chain DJ
rearrangements (162b). The finding that IL-
7 effectively suppresses myeloid differentia-
tion strongly argues for an instructive effect of
this cytokine. Pax5 is also required through-
out B cell development to maintain commit-
ment. Thus, conditional deletion in B cells
caused the downregulation of B cell markers
and derepression of myeloid genes even in ma-
ture B cells (164); after transfer to appropri-
ate conditions, these cells differentiated into
macrophages (165). How Pax5 represses the
expression of various myelomonocytic genes,
such as that of M-CSFR (166) and Notch1 (96),
remains to be determined.
Similar observations were made with mice
defective in the E2A gene (Figure 9). This
gene regulates expression of EBF, which in
turn regulates the B cell commitment factor
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Pax5 (63). B cell development in E2A
is blocked at the pro-B cell stage (85, 109,
167). Cultured E2A
pro-B cells express
multiple myeloid genes (168), which likely
results from the absence of Pax5 (95). Im-
portantly, like Pax5
cells, E2A
were capable of differentiating in vivo into all
hematopoietic lineages tested (with the excep-
tion of B cells), as shown by transplantation
into irradiated hosts (168). However, whether
freshly isolated E2A
cells not selected in
culture are capable of multilineage differenti-
ation is unclear. That E2A
cells also gener-
ate T cells is surprising given the deficiency in
T cell formation from progenitors of E2A
mice (109). This raises the possibility that ex-
pression of a redundant factor abrogates the
requirement for E2A in T cell differentiation.
PU.1 is another example of a transcrip-
tion factor whose ablation induces lineage-
inappropriate gene expression. Cultured
fetal liver–derived B cells lacking PU.1 ac-
Figure 10
Asymmetries in lymphoid and myeloid cell plasticity. Scheme
summarizing experiments showing reprogramming of CLPs into MegE
cells by GATA-1 (145) and into GM cells by C/EBP
expression (M.
Kondo, personal communication). In addition, the scheme illustrates
reprogramming of pro-B, pre-B, pro-T, and pre-T cells into macrophages
by enforced expression of C/EBP
(154; C.V. Laiosa, H. Xie & T. Graf,
unpublished results).
tivate the expression of a subset of T cell
genes, including ZAP-70 and lck (84a). There-
fore, the apparent requirement of PU.1 down-
regulation at the pro-T/pre-T cell transition
for T cell commitment (23, 114) might be
explained by the ability of PU.1 to repress
T cell genes. In addition, inactivation or re-
duction of PU.1 levels in B lineage cells in
vivo induced a dramatic shift from B-2 to B-
1 cells (84a, 168a). An unsuspected link to
leukemia was observed in animals in which
alterations or deletions of a regulatory site
within the PU.1 enhancer, leading to a re-
duced expression of the factor, induced acute
myeloid and T cell leukemia (168a, 169).
PU.1 dosage also influences lymphoid ver-
sus myeloid specification because enforced
expression of PU.1 in PU.1
fetal liver
progenitors showed that higher levels of the
protein favored myelomonocytic specification
rather than B cell formation (170). However,
as mentioned above, it is unclear whether
this is a true dosage effect because under
certain culture conditions B cells develop
in a PU.1-independent manner, as for fe-
tal liver progenitors. The multiple functions
of PU.1 in the differentiation and prolif-
eration of both lymphoid and myeloid lin-
eages show that this transcription factor has
a prominent role as a master coordinator of
Why can the enforced expression of a single
transcription factor reprogram lymphoid-to-
myeloid cells, whereas lymphoid-to-myeloid
conversions have not been achieved (sum-
marized in Figure 10)? A simple explana-
tion is that the conversions have not been
achieved owing to technical inadequacies of
the experiments performed so far. Another
is that the plasticity bias reflects irreversible
chromatin changes in myeloid cells that
724 Laiosa
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ANRV270-IY24-23 ARI 23 February 2006 15:47
prevent accessibility of lymphoid genes to
lymphoid transcription factors. However, the
data discussed in this review suggest an alter-
native explanation. First, a comparison of the
myeloid and lymphoid compartments shows
striking differences in their transcription fac-
tor network complexities. Thus, as described
above, at least four transcription factors are
required for B and T lineage establishment,
whereas two appear to be sufficient to spec-
ify myeloid lineages. Second, the B cell tran-
scription factor network is set up sequentially
and strengthened by external inputs, such as
signaling through Flt3 and IL-7R (2). Al-
though somewhat less clear, the T cell tran-
scription factor network is possibly also set
up in a sequential fashion and depends on
signaling through the Notch1 receptor (23,
62). In contrast, the simple binary transcrip-
tion factor code of myeloid cells can be es-
tablished in one or two steps that might be
independent of external stimuli. Third, re-
lated to the binary code of myeloid cells, at
least some myeloid instructive transcription
factors can establish feed-forward loops, up-
regulating their respective partner. In con-
trast, lymphoid transcription factors do not
appear to do so or, if they do, not in a recip-
rocal fashion. For example, although EBF can
activate Pax5 expression, the reciprocal mech-
anism, Pax5-activating expression of EBF, is
not known to occur. The notion that complex
transcription factor combinations, assembled
in a sequential manner, specify lymphoid cells,
whereas much simpler combinations spec-
ify myeloid cells, may reflect their more
recent evolutionary origin (18). Thus, the ba-
sic wiring of myeloid cells may have served
as a platform for the design of more com-
plex cell phenotypes. This idea is also sup-
ported by the fact that B and T cell lineages
appear relatively late during ontogeny (170a)
and that they exhibit a number of well-defined
developmental stages, not seen in myeloid lin-
eages (171–173). The above speculations pre-
dict that to achieve the reprogramming of
myeloid into lymphoid lineages or lymphoid
into other lymphoid lineages will require the
Lineage priming:
expression of
genes in multipotent
expression of two or more lymphoid instruc-
tive transcription factors, perhaps in a defined
Commitment of HSCs and progenitor cells
to a given lineage may simply be caused by
the activation of one or several key tran-
scription factors that then establish the gene
expression profile characteristic of, for ex-
ample, a macrophage or a B cell. Thus,
HSCs might start out as blank slates that ex-
press no genes characteristic of differentiated
cells. However, Enver and colleagues (174)
showed that single multipotent hematopoi-
etic cells coexpress genes at low levels nor-
mally found in mature MegE and GM cells,
such as hemoglobin and myeloperoxidase.
Subsequent studies have shown that lineage
priming or lineage promiscuity occurs in all
adult hematopoietic progenitor cells, includ-
ing long-term HSCs (175, 176) and in MPPs
isolated from the early mouse embryo (177).
The coexistence of different transcriptional
programs in progenitor cells, followed by the
stepwise extinction of all but one of them, is
therefore a defining feature of the hematopoi-
etic system.
The repertoire of primed genes is not the
same in different hematopoietic progenitor
cells. Thus, single long-term HSCs as well as
CMPs coexpress a wide variety of markers of
mature GM and MegE cells, including several
myeloid cytokine receptors as well as the tran-
scription factors PU.1, C/EBP
, and GATA-
1. GMPs and MEPs, in turn, only express
GM- or MegE-associated genes, respectively.
As expected, CLPs prime both T and B cell
genes but not myeloid genes (175). Priming
therefore appears to be restricted to genes ex-
pressed in the physiological progeny of a cell.
However, markers of mature B and T cells
are essentially absent from long-term HSCs
(175), which is also evidenced by the total
lack of myeloid cell labeling in a mouse model
Lymphoid-Myeloid Development 725
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ANRV270-IY24-23 ARI 23 February 2006 15:47
that traces the progeny of CD19-expressing
cells in vivo (176). Of the specific regulators
of lymphoid development, only GATA-3 and
Notch1, both implicated in T cell develop-
ment, are expressed in long-term HSCs, al-
though the Notch1 target gene pT
cannot be
detected (175, 178, 179). The gene expression
program of lymphocytes is therefore not pre-
viewed in HSCs, in contrast to that of myeloid
Because HSCs express myeloid markers as
well as several of the key myeloid regula-
tors, but appear to express no lymphoid mark-
ers and few T cell–associated transcription
factors, they resemble more closely myeloid
than lymphoid progenitors. The expression
of lineage-restricted markers in early pro-
genitors is most likely a direct reflection of
the transcription factor combinations they ex-
press. A summary of lineage-instructive tran-
scription factors expressed in HSCs as well as
in selected intermediate and monopotent pro-
genitors is shown in Figure 11. The scheme
supports the notion that lineage specification
involves a stepwise simplification of transcrip-
tion factor networks, with fully committed
cells representing stable, low-energy states.
That the transcription factor network com-
plexity of CLPs appears to be higher suggests
that HSCs have to go uphill to become lym-
phoid. This notion sharply focuses the ques-
tion as to how HSCs escape a myeloid fate and
instead become lymphoid.
In light of the self-renewal and multilin-
eage differentiation capacities of HSCs, one
must assume that the expression of myeloid-
instructive factors is too low to induce myeloid
commitment, at least in the bulk of the popu-
lation. Small transcription factor fluctuations
and imbalances in HSCs might then repre-
sent the first step in lineage commitment.
Transcription factor antagonisms and syner-
gisms, combined with external signals such
as cytokine receptor signaling, could amplify
such imbalances, making commitment irre-
versible. Although whether lineage priming
in HSCs is an oscillatory process has not yet
been determined, RT-PCR analysis suggests
that HSCs alternate between different pat-
terns of gene expression (175), some of which
might be more conducive for commitment
to a certain lineage than others. However, at
least for the expression of the myeloid marker
lysozyme, a developmental bias based on lin-
eage priming does not exist because CMPs ex-
pressing lysozyme M differentiate into MegE
cells at similar frequencies (175). Neverthe-
less, this situation could be different for the
stochastic upregulation of cytokine receptors,
which then can be triggered by environmental
Important players in the loss of self-
renewal, entry into cell cycle, and initiation of
lymphoid differentiation of HSCs/MPPs may
be the tyrosine kinase receptor Flt3 and the
transmembrane receptor Notch1. Thus, on
the one hand, expression of Flt3, which causes
myeloid leukemias when mutated (180), is
progressively upregulated from long-term
HSCs, to short-term HSCs, to MPPs, cor-
relating with the loss of self-renewal potential
(9). On the other hand, Flt3 is required for
the generation of CLPs (53), and its ligand-
mediated activation in synergy with PU.1 may
induce the expression of IL-7R
which in turn
upregulates EBF (58a) in cells destined to be-
come B cells (2). Similarly, Notch1, which is
frequently mutated in T cell leukemias (181),
is progressively upregulated from long-term
HSCs to MPPs (178). This upregulation may
be important for the successful seeding of
the thymus and T cell specification. In con-
trast to the expression pattern of Flt3 and
Notch1, GATA-1 and GATA-2 are expressed
at higher levels in long-term HSCs than in
short-term HSCs or MPPs (178). The ap-
parent inverse regulation of myeloid- and
lymphoid-associated regulators in LSK cells
raises the possibility that they inhibit each
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ANRV270-IY24-23 ARI 23 February 2006 15:47
prpro-T o-B
Figure 11
Transcription factor network complexity of HSCs, intermediate, and monopotent progenitors. The
expression of lineage-instructive transcription factors is indicated within each cell type. The levels of a
given transcription factor may differ substantially in the various cell types shown, and this may be of
biological significance. GATA-2, which is expressed in all intermediate myeloid progenitors, is not
included because its significance in establishing lineage-specific gene expression programs is unclear.
(References used to compile the factors listed: 14, 35, 58, 72, 175, 178, 194.)
other, changing probabilities of lymphoid ver-
sus myeloid lineage specification.
As highlighted by this review, a great deal
has been learned about the determinants that
specify lymphoid and myeloid fate, and in
particular about the role of transcription fac-
tors in these processes. However, many open
questions remain, such as whether lymphoid-
myeloid branching occurs at invariant po-
sitions along differentiation pathways (as
proposed by the CMP/CLP model) or occurs
with certain probabilities within the progeny
of different early hematopoietic progenitors.
Although the reprogramming experiments
discussed provide plausible scenarios about
how lymphoid cells diverge from myeloid cells
and why they retain a certain degree of plastic-
ity, future experiments must address this issue
in a physiological context. One problem that
was brought into sharp focus is how HSCs,
which more closely resemble myeloid than
lymphoid precursors, acquire a lymphoid fate.
We hope that this interpretation of the avail-
able data will help in designing experiments
that directly approach this question.
Lymphoid-Myeloid Development 727
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ANRV270-IY24-23 ARI 23 February 2006 15:47
We thank Emmanuelle Passegue, Cornelius Murre, Daniel Tenen, Arthur Skoultchi, and
Motonari Kondo for providing unpublished data; Ellen Rothenberg, Juan Carlos Zuniga-
Pflucker, and Fabio Rossi for comments on the manuscript; and Huafeng Xie for helpful
discussions as well as contribution of the data analysis shown in Figure 7. This work was
supported by NIH grants R01 CA89590-01 and R01 NS43881-01.
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Contents ARI 7 February 2006 11:41
Annual Review
of Immunology
Volume 24, 2006
Jack L. Strominger ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp x
The Tortuous Journey of a Biochemist to Immunoland and What He
Found There
Jack L. Strominger ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp1
Osteoimmunology: Interplay Between the Immune System and Bone
Matthew C. Walsh, Nacksung Kim, Yuho Kadono, Jaerang Rho, Soo Young Lee,
Joseph Lorenzo, and Yongwon Choi pppppppppppppppppppppppppppppppppppppppppppppppppppppppp33
A Molecular Perspective of CTLA-4 Function
Wendy A. Teft, Mark G. Kirchhof, and Joaquín Madrenas pppppppppppppppppppppppppppppppp65
Transforming Growth Factor-β Regulation of Immune Responses
Ming O. Li, Yisong Y. Wan, Shomyseh Sanjabi, Anna-Karin L. Robertson,
and Richard A. Flavell ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp99
The Eosinophil
Marc E. Rothenberg and Simon P. Hogan ppppppppppppppppppppppppppppppppppppppppppppppppp147
Human T Cell Responses Against Melanoma
Thierry Boon, Pierre G. Coulie, Benoît J. Van den Eynde,
and Pierre van der Bruggen ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp175
FOXP3: Of Mice and Men
Steven F. Ziegler ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp209
HIV Vaccines
Andrew J. McMichael ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp227
Natural Killer Cell Developmental Pathways: A Question of Balance
James P. Di Santo ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp257
Development of Human Lymphoid Cells
Bianca Blom and Hergen Spits ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp287
Genetic Disorders of Programmed Cell Death in the Immune System
Nicolas Bidère, Helen C. Su, and Michael J. Lenardo ppppppppppppppppppppppppppppppppppppp321
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Contents ARI 7 February 2006 11:41
Genetic Analysis of Host Resistance: Toll-Like Receptor Signaling and
Immunity at Large
Bruce Beutler, Zhengfan Jiang, Philippe Georgel, Karine Crozat, Ben Croker,
Sophie Rutschmann, Xin Du, and Kasper Hoebe ppppppppppppppppppppppppppppppppppppppp353
Multiplexed Protein Array Platforms for Analysis of Autoimmune
Imelda Balboni, Steven M. Chan, Michael Kattah, Jessica D. Tenenbaum,
Atul J. Butte, and Paul J. Utz pppppppppppppppppppppppppppppppppppppppppppppppppppppppppp391
How TCRs Bind MHCs, Peptides, and Coreceptors
Markus G. Rudolph, Robyn L. Stanfield, and Ian A. Wilson ppppppppppppppppppppppppppppp419
B Cell Immunobiology in Disease: Evolving Concepts from the Clinic
Flavius Martin and Andrew C. Chan pppppppppppppppppppppppppppppppppppppppppppppppppppppp467
The Evolution of Adaptive Immunity
Zeev Pancer and Max D. Cooper ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp497
Cooperation Between CD4
and CD8
T Cells: When, Where,
and How
Flora Castellino and Ronald N. Germain pppppppppppppppppppppppppppppppppppppppppppppppppp519
Mechanism and Control of V(D)J Recombination at the
Immunoglobulin Heavy Chain Locus
David Jung, Cosmas Giallourakis, Raul Mostoslavsky,
and Frederick W. Alt ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp541
A Central Role for Central Tolerance
Bruno Kyewski and Ludger Klein pppppppppppppppppppppppppppppppppppppppppppppppppppppppppp571
Regulation of Th2 Differentiation and Il4 Locus Accessibility
K. Mark Ansel, Ivana Djuretic, Bogdan Tanasa, and Anjana Rao ppppppppppppppppppppppp607
Diverse Functions of IL-2, IL-15, and IL-7 in Lymphoid Homeostasis
Averil Ma, Rima Koka, and Patrick Burkett pppppppppppppppppppppppppppppppppppppppppppppp657
Intestinal and Pulmonary Mucosal T Cells: Local Heroes Fight to
Maintain the Status Quo
Leo Lefrançois and Lynn Puddington ppppppppppppppppppppppppppppppppppppppppppppppppppppppp681
Determinants of Lymphoid-Myeloid Lineage Diversification
Catherine V. Laiosa, Matthias Stadtfeld, and Thomas Graf pppppppppppppppppppppppppppppp705
GP120: Target for Neutralizing HIV-1 Antibodies
Ralph Pantophlet and Dennis R. Burton ppppppppppppppppppppppppppppppppppppppppppppppppppp739
Compartmentalized Ras/MAPK Signaling
Adam Mor and Mark R. Philips ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp771
vi Contents
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... 3 In mice, nonphagocytic cells of the lymphoid and erythroid lineages have retained the potential to convert into macrophages, 4,5 suggesting that the myeloid lineage represents a default pathway. 6 These observations raise several questions: Is there an evolutionarily conserved gene expression program that determines phagocytic capacity? If so, when did it evolve and is it driven by an ancestral transcription factor(s)? ...
... They report that tunicate-and spongederived phagocytes, as well as Capsaspora cells, show higher transcriptional similarities with macrophages than nonphagocytic cells of mice and also selectively express high levels of the gene encoding the transcription factor CEBPα. Strikingly, Cebpa was also found to be expressed among a small number of genes conserved in Capsaspora, along with the lysosome/ phagocytosis-associated gene Pla2g15. It is known that CEBPα plays an important role in myeloid cell specification, that its overexpression in Band T-cell precursors induces the conversion into functional macrophages, 6 and that it is also involved in the specification and function of a number of additional cell types, including adipocytes and hepatocytes. 7 Consequently, the authors tested the macrophage-inducing capacity of the CEBPα homologs from Ciona, Amphimedon, and Capsaspora in mouse lymphoid and megakaryocytic precursors. ...
... Remarkably, similar findings were made in vivo since 6 raising the possibility that this gene is less conserved than Cebpa or that another cooperating factor(s) is yet to be found. More fundamentally, the findings described support the idea that primitive phagocytes are evolutionary ancestors of not only myeloid/lymphoid and erythroid/megakaryocytic cells but also HSCs. ...
... 42 In addition, it is worth mentioning that cell fate plasticity and transcription factor-mediated lineage conversion have been extensively reported for the B cell but not the T cell compartment. 43 44 The absence of evident immune escape to either CD1a-CAR or CD1a-STAb T cells may explain the very similar efficacy in controlling leukemia progression in multiple in vivo models despite the apparently more potent and rapid in vitro cytotoxic responses of CD1a-STAb T cells. In summary, CD1a-STAb T therapy could be an alternative to CD1a-CAR T in T-ALL, especially in R/R patients with leukapheresis products showing limited numbers of nontumoral effector T cells. ...
Full-text available
Background The dismal clinical outcome of relapsed/refractory (R/R) T cell acute lymphoblastic leukemia (T-ALL) highlights the need for innovative targeted therapies. Although chimeric antigen receptor (CAR)-engineered T cells have revolutionized the treatment of B cell malignancies, their clinical implementation in T-ALL is in its infancy. CD1a represents a safe target for cortical T-ALL (coT-ALL) patients, and fratricide-resistant CD1a-directed CAR T cells have been preclinically validated as an immunotherapeutic strategy for R/R coT-ALL. Nonetheless, T-ALL relapses are commonly very aggressive and hyperleukocytic, posing a challenge to recover sufficient non-leukemic effector T cells from leukapheresis in R/R T-ALL patients. Methods We carried out a comprehensive study using robust in vitro and in vivo assays comparing the efficacy of engineered T cells either expressing a second-generation CD1a-CAR or secreting CD1a x CD3 T cell-engaging Antibodies (CD1a-STAb). Results We show that CD1a-T cell engagers bind to cell surface expressed CD1a and CD3 and induce specific T cell activation. Recruitment of bystander T cells endows CD1a-STAbs with an enhanced in vitro cytotoxicity than CD1a-CAR T cells at lower effector:target ratios. CD1a-STAb T cells are as effective as CD1a-CAR T cells in cutting-edge in vivo T-ALL patient-derived xenograft models. Conclusions Our data suggest that CD1a-STAb T cells could be an alternative to CD1a-CAR T cells in coT-ALL patients with aggressive and hyperleukocytic relapses with limited numbers of non-leukemic effector T cells.
... A critical balance between HSC self-renewal and differentiation into different hematopoietic lineages must be maintained throughout an individual's life in order to maintain an effective immune system and normal oxygen transport. As with many biological systems, transcriptional regulations orchestrated by transcription factors (TFs) and their networking are key mechanisms for instructing differentiation occurring in the hematopoietic stem and progenitor cell (HSPC) compartment (reviewed in [1,2]). The characterization of their activities has led to refine our view of the multiple branch points for early hematopoiesis [3]. ...
Full-text available
Hematopoietic stem cell (HSC) aging is a multifactorial event leading to changes in HSC properties and functions, which are intrinsically coordinated and affect the early hematopoiesis. To better understand the mechanisms and factors controlling these changes, we developed an original strategy to construct a Boolean model of HSC differentiation. Based on our previous scRNA-seq data, we exhaustively characterized active transcription modules or regulons along the differentiation trajectory and constructed an influence graph between 15 selected components involved in the dynamics of the process. Then we defined dynamical constraints between observed cellular states along the trajectory and using answer set programming with in silico perturbation analysis, we obtained a Boolean model explaining the early priming of HSCs. Finally, perturbations of the model based on age-related changes revealed important deregulations, such as the overactivation of Egr1 and Junb or the loss of Cebpa activation by Gata2. These new regulatory mechanisms were found to be relevant for the myeloid bias of aged HSC and explain the decreased transcriptional priming of HSCs to all mature cell types except megakaryocytes.