ne w s and v ie w s
particular subunits of RNA polymerase III whether RNA isolated from the nucleus of
as being important for the recognition and
transcription of dsDNA. Chiu et al. isolate
RNA from the cytosol and nucleus and show
that only cytosolic RNA induces IFN-β pro-
duction. This finding is consistent with the
idea that cellular RNA transcribed in the
nucleus is further processed to remove or
modify (for example, cap) 5′-triphosphate
before transport to the cytoplasm and thus
loses the ability to activate the interferon
pathway. Although both studies find that the
interferon-inducing activity of RNA isolated
from cells transfected with dsDNA is located
mainly in the cytosol, it is still not clear
A defining factor for natural killer cell development
© 2009 Nature America, Inc. All rights reserved.
James P Di Santo
Transcription factors are critical regulators of cell fate in the hemato-lymphoid system. New evidence indicates
that the basic leucine zipper transcription factor E4BP4 (also known as NFIL3) is essential for natural killer cell
specification.
L ymphocyte development from
hematopoietic stem cells is a carefully
orchestrated process that integrates signals
(both cell-associated, membrane-bound
factors and soluble factors) elaborated by
stromal cells in the bone marrow and thy-
mic microenvironments. These extrinsic
signals converge in the nuclei of respond-
ing hematopoietic precursors and regulate
a multitude of transcriptional targets nec-
essary to guide the cells along distinct dif-
ferentiation pathways leading to lymphoid
precursors and later to fully differentiated
B cells, T cells or natural killer (NK) cells.
Central to this process are a variety of tran-
scription factors that have the ability to
form higher-ordered, multiprotein com-
plexes that interact with DNA and modify
the transcriptional activity of particular
target genes. ‘Master’ transcription factors
essential for the development of B cells and
T cells (including αβ T cells, γδ T cells and
NKT cells) have been identified, which has
provided key information about how these
lymphoid subsets are generated (Fig. 1). In
contrast, a defining factor for NK cell devel-
James P. Di Santo is in the Cytokines and Lymphoid
Development Unit, Institut Pasteur, and Institut
National de la Santé et de la Recherche Médicale
U668, Paris, France.
e-mail: disanto@pasteur.fr
nature immunology volume 10 number 10 october 2009
1. Takeuchi, O. & Akira, S. Immunol. Rev. 227, 75–86
target cells infected with DNA virus or bac-
teria would be similarly devoid of interferon-
inducing activity.
Finally, are any unknown factors or cofac-
tors required for cytosolic RNA polymerase
III–mediated sensing and transcription of
foreign DNA? Are there additional cytosolic
DNA sensors that remain to be discovered?
The finding that induction of type I inter-
feron by dsDNA in mouse cells does not
depend on the RIG-I pathway suggests the
presence of another sensor for direct recog-
nition of foreign DNA to trigger the produc-
tion of type I interferon.
NKP
HSC ELP TSP
Nonlymphoid pro-B
lineages
Figure 1 Transcription factor control of lymphoid lineage development. The key transcription
factors E4BP4, Id2, Notch, GATA-3, EBF, Pax5, Th-POK, PLZF and SOX13 have been identified as
being essential for the development of various lymphoid cell types. HSC, hematopoietic stem cell;
ELP, early lymphoid precursor; TSP, thymus-settling cell; pro-B, pro–B cell; pro-T, pro–T cell;
B, B cell; T, T cell.
opment has remained elusive, and it has
remained possible that NK cell development
represents a ‘default pathway’ used when
other lymphoid lineages are not directly
specified. In this issue of Nature Immunology,
Brady and colleagues put this issue to rest
by identifying E4BP4 (E4-binding protein 4;
also known as NFIL3 (nuclear factor inter-
leukin 3 (IL-3) regulated)), a basic leucine
zipper (bZIP) transcriptional activator, as
a critical transcription factor required for
NK cell development 1. These landmark
observations provide a framework for fur-
ther delineation of the processes involved in
NK cell lineage commitment and indicate
involvement of this bZIP-family transcrip-
tion factor in the differentiation of NK cells
(2009).
2. Takaoka, A. et al. Nature 448, 501–505 (2007).
3. Schroder, K., Muruve, D.A. & Tschopp, J. Curr. Biol.
19, R262–R265 (2009).
4. Ablasser A. et al. Nat. Immunol. 10, 1065–1072
(2009).
5. Chiu, Y.H., Macmillan, J.B. & Chen, Z.J. Cell 138,
576–591 (2009).
6. Cheng, G., Zhong, J., Chung, J. & Chisari, F.V. Proc.
Natl. Acad. Sci. USA 104, 9035–9040 (2007).
7. Ishii, K.J. et al. Nat. Immunol. 7, 40–48 (2006).
8. Opitz, B. et al. J. Biol. Chem. 281, 36173–36179
(2006).
9. Yoshida, H., Okabe, Y., Kawane, K., Fukuyama, H. &
Nagata, S. Nat. Immunol. 6, 49–56 (2005).
10. Ronnblom, L. & Pascual, V. Lupus 17, 394–399
(2008).
11. Samanta, M., Iwakiri, D., Kanda, T., Imaizumi, T. &
Takada, K. EMBO J. 25, 4207–4214 (2006).
E4BP4
Id2
NK
O K
CD4+ T
Th-P
Notch1 PLZF
pro-T NKT
GATA-3 SO
X1
3
EBF
B γδ T
Pax5
(and perhaps other lymphocytes with innate
characteristics).
E4BP4 was initially identified by its abil-
ity to repress viral transcription and to
promote IL-3-dependent survival of pro–B
cells2. Subsequent molecular characteriza-
tion has demonstrated that E4BP4 contains
a canonical bZIP motif, which places this
transcription factor in the extended bZIP
protein family that includes several hun-
dred members in different species, includ-
ing CREB, ATF, AP1 and C/EBP proteins;
E4BP4 belongs to the proline- and acid-
rich subfamily2. The bZIP proteins are
critically involved in the control of diverse
developmental processes in and outside the
hematopoietic system, which suggests E4BP4
1051
 ne w s and v ie w s
might serve a role during hemato-lymphoid
development. Although E4BP4-deficient
mice (Nfil3–/–; called ‘E4bp4–/–’ here) develop
without apparent abnormalities and have
an essentially normal hematopoietic and
lymphoid profile, mature NK cells (identi-
fied as CD3–NKp46+NK1.1+) are conspicu-
ously absent from the bone marrow, spleen
and blood. Furthermore, classical NK cell
functions (such as elimination of major his-
tocompatibility complex class I–deficient
target cells and production of interferon-γ)
are not detected in E4bp4–/– mice, whereas
influenza-specific antiviral CD8 + T cell
responses are intact, which suggests a severe
and selective NK cell deficiency. Using irra-
diated bone marrow chimeras, Gascoyne
et al. show that hematopoietic expression of
E4BP4 is required for normal NK cell devel-
© 2009 Nature America, Inc. All rights reserved.
opment1; this is important, as E4BP4 expres-
sion outside the hematopoietic system2 could
have conditioned a lymphopoietic microen-
vironmental niche critical for NK cell gen-
eration. They also find that ectopic retroviral
expression of E4BP4 in hematopoietic stem
cells promotes NK cell development in vitro
and partially corrects the NK cell deficiency
of E4bp4–/– mice in vivo1. Collectively, these
studies identify E4BP4 as the first transcrip-
tion factor known to be selectively and criti-
cally required for NK cell development.
As previous studies have indicated involve-
ment of E4BP4 in growth factor–dependent
lymphocyte survival, Gascoyne et al. go on
to investigate a possible link between E4BP4
and soluble factors known to be involved in
NK cell development. ‘Trans-presentation’ of
IL-15 by membrane-bound IL-15 receptor-α
represents the essential ‘fuel’ that drives the
differentiation of immature NK cells (iNK
cells) from committed NK cell precursors
(NKPs) and maintains the homeostasis of
mature peripheral NK cells3. E4BP4 expres-
sion is detected in NKPs, is upregulated in
immature NK cells and is maintained in
mature NK cells. This expression pattern
correlates with the IL-15 dependency of
these different stages of NK cell develop-
ment4 and suggests a model in which E4BP4
is a critical transcriptional target that medi-
ates the biological effects of IL-15. In sup-
port of this view, Gascoyne et al. show that
IL-15 enhances E4BP4 expression in IL-15-
responsive target cells and that ectopic
expression of E4BP4 allows some degree of
NK cell development from hematopoietic
stem cells when IL-15 signaling is blocked1.
Interestingly, the positive effects of E4BP4 on
NK cell homeostasis do not seem to involve
improved survival or proliferation, leading
Gascoyne et al. to propose that E4BP4 acts
1052
mainly by promoting NK lineage commit-
ment. Although the authors do not formally
address this hypothesis, the fact that E4BP4
is required at the earliest stage of NK cell
development (E4bp4–/– mice have normal
numbers of NKPs but considerably fewer
immature and mature NK cells) is consistent
with a role for E4BP4 in the specification of
NK cells. As NKPs comprise a heterogeneous
population (only 10% give rise to NK cells
in vitro5), E4BP4 expression may provide a
more precise marker for the identification of
true NKPs as well as the extracellular triggers
that lead to their specification.
What are the molecular targets of E4BP4
transcriptional activation? Here, Brady and
colleagues analyze E4BP4-sufficient and
E4BP4-deficient hematopoietic precursors
and show that expression of the basic helix-
loop-helix inhibitor Id2 is much lower in
the absence of E4BP4. Id2 has been shown
to serve a critical role in NK cell homeosta-
sis (Id2–/– mice have much lower NK cell
numbers than wild-type mice6) by oppos-
ing the activity of E-box transcription fac-
tors, which strongly promote B cell and T cell
lymphopoiesis7. Gascoyne et al. show that
E4BP4 induces Id2 expression and that ecto-
pic Id2 expression partially restores NK cell
development in vitro from E4BP4-deficient
hematopoietic precursors1. These data are
consistent with a transcription factor cascade
in which E4BP4 induces Id2 expression in
early lymphoid precursors, which can then
promote the NK cell fate8.
Id2 has critical developmental roles beyond
its ability to promote NK cell development9. In
the hematopoietic system, Id2 has an essential
role in the differentiation of dendritic cells,
osteoclasts and lymphoid tissue-inducer cells,
a population of CD3–CD4+CD117+CD127+
cells that constitutively express the gene
encoding the transcription factor RORγt and
are required for the development of prepro-
grammed and induced lymphoid structures,
including Peyer’s patches, lymph nodes and
intestinal cryptopatches10. Given a model
in which E4BP4 controls Id2 expression, it
would be expected that the phenotypes of
E4bp4–/– mice and Id2–/– mice would con-
verge. However, this does not seem to be the
case, as E4bp4–/– mice have normal develop-
ment of lymph nodes and Peyer’s patches
(H. Brady, personal communication), which
suggests that lymphoid tissue–inducer activ-
ity is largely intact in the absence of E4BP4.
Nevertheless, these observations point to a
cell type–specific control of Id2 expression
in the hematopoietic system that may be
achieved through transcriptional activation
by alternative members of the bZIP family or
volume 10 number 10 october 2009 nature immunology
by other transcription factors that can com-
pensate for E4BP4.
The notion that E4BP4 may be redundant
for the activation of certain transcriptional
targets in some cell types may also explain
the lack of any obviously altered phenotype
of NKT cells and memory CD8+ T cells in
E4bp4–/– mice. Like NK cells, these cell types
show constitutive E4BP4 expression that can
be enhanced by IL-15, but their homeostasis
is unaffected by the absence of E4BP4 (ref. 1).
One explanation for this difference in sensitiv-
ity might involve enhanced responsiveness of
these T cell subsets to related growth factors.
In this context, another common γ-chain–
dependent cytokine (IL-7) has been shown
to compensate for IL-15 in memory T cell
homeostasis11. Still, a more complete analy-
sis of NKT and memory CD8+ T cell function
in E4bp4–/– mice is warranted before the pos-
sibility of a role for E4BP4 in the maturation
of these cells is excluded. In a similar fashion,
although studies of E4bp4–/– mice have identi-
fied a critical role for E4BP4 during early NK
cell development, conditional ablation studies
will be needed to address whether persistent
E4BP4 expression is required for maintenance
of NK cell function.
Finally, E4bp4–/– mice may provide a new
tool for studying diurnal regulation in the
immune system. Published studies of droso-
phila and chickens have suggested a role for
E4BP4 homologs in the regulation of cir-
cadian ‘clocks’2. These ‘clocks’ are activity
rhythms that oscillate intrinsically but are
synchronized by environmental cues and are
controlled by autoregulatory transcription
and translation feedback loops 12. Diurnal
regulation of innate and adaptive immune
function is an emerging field 13, and the
identification of E4BP4 expression in innate
lymphocytes could provide a means of sub-
stantiating molecular ‘clock’ mechanisms in
these cells of the innate immune response.
1. Gascoyne, D.M. et. al, Nat. Immunol. 10, 1118–1124
(2009)
2. Cowell, I.G. Bioessays 24, 1023–1029 (2002).
3. Di Santo, J.P. Annu. Rev. Immunol. 24, 257–286
(2006).
4. Vosshenrich, C.A. et al. J. Immunol. 174, 1213–1221
(2005).
5. Rosmaraki, E.E. et al. Eur. J. Immunol. 31, 1900–1909
(2001).
6. Boos, M.D., Yokota, Y., Eberl, G. & Kee, B.L. J. Exp.
Med. 204, 1119–1130 (2007).
7. Yokota, Y. et al. Nature 397, 702–706 (1999).
8. Fujimoto, S., Ikawa, T., Kina, T. & Yokota, Y. Int.
Immunol. 19, 1175–1182 (2007).
9. Yokota, Y. Oncogene 20, 8290–8298 (2001).
10. Eberl, G. Trends Immunol. 28, 423–428 (2007).
11. Kieper, W.C. et al. J. Exp. Med. 195, 1533–1539
(2002).
12. Hastings, M., O’Neill, J.S. & Maywood, E.S.
J. Endocrinol. 195, 187–198 (2007).
13. Arjona, A. & Sarkar, D.K. Neurochem. Res. 33, 708–
718 (2008).