Immunity, Vol. 21, 43–53, July, 2004, Copyright 2004 by Cell Press
Plasmacytoid Dendritic Cells
Activate Lymphoid-Specific Genetic Programs
Irrespective of Their Cellular Origin
2002). In murine hematopoiesis, early studies showed
that CD11c?MHCII?CD11b?CD8??“myeloid” DCs can
be efficiently derived from myeloid progenitors along
with granulocytes and macrophages (Inaba et al., 1993),
whereas CD11c?MHCII?CD11b?CD8??“lymphoid” DCs
can arise from thymic T cell progenitors (Ardavin et al.,
1993). Further analyses revealed that DCs of these two
phenotypes can originate both from the earliest myeloid
progenitors (common myeloid progenitors, CMPs [Akashi
et al., 2000]) and from the earliest lymphoid progenitors
(common lymphoid progenitors, CLPs [Kondo et al.,
1997]) (Traver et al., 2000). The potential to develop
into the DC lineage is maintained in pro-T cells and
(MEPs) (Manz et al., 2001; Wu et al., 2001), although
some DC potential may be retained in more primitive B
cell precursors (Izon et al., 2001). Thus, DC develop-
monocyte and T lymphoid developmental pathways.
Plasmacytoid dendritic cells (PDCs) were initially
identified in humans (Cella et al., 1999; Grouard et al.,
These cells morphologically resemble antibody-secre-
activation, they quickly acquire mature DC morphology
and phenotype without significant cell proliferation (Liu,
2001; Shortman and Liu, 2002). PDCs secrete massive
amounts of type I interferons (IFN-? and -?) when stimu-
lated by unmethylated CpG DNA or by viruses (Cella et
al., 1999; Grouard et al., 1997; Jarrossay et al., 2001;
Kadowaki et al., 2000, 2001; Siegal et al., 1999) through
the function of Toll-like receptors such as TLR7 and
TLR9 (Kadowaki et al., 2001) and are thus believed to
also play a role in adaptive immunity by directing either
T helper cell type 1 (Th1) or Th2 development (Boonstra
et al., 2003), as well as by generating T regulatory cells
that induce T cell tolerance (Gilliet and Liu, 2002). Re-
cently, a murine counterpart of human PDCs has been
ties (Asselin-Paturel et al., 2001; Bjorck, 2001; Martin et
al., 2002; Nakano et al., 2001). Like human PDCs, mouse
PDCs express high levels of CD45RA (B220) and low
levels of MHC class II (MHCII). Mouse PDCs express
low levels of the dendritic cell marker CD11c, and the
majority of them are CD8??. In addition, mouse PDCs
are Ly-6C?, a myeloid cell marker recognized by the
Gr-1 antibody that also reacts with Ly-6G on mature
granulocytes and monocytes.
The lineage origin of PDCs is controversial. On the
one hand, murine PDCs have been reported to carry
rearranged immunoglobulin heavy chain (IgH) D-J but
not T cell receptor (TCR) D?-J? loci (Corcoran et al.,
2003). Another apparently lymphoid characteristic of
PDCs is the expression of the pre-T cell receptor ? (pT?)
in the human thymic PDCs (Bendriss-Vermare et al.,
2001; Res et al., 1999). The pT? protein pairs with the
TCR? chain to form the pre-TCR (Groettrup et al., 1993)
Hirokazu Shigematsu,1,6Boris Reizis,2,6,7
Hiromi Iwasaki,1Shin-ichi Mizuno,1Dan Hu,1
David Traver,3Philip Leder,2Nobuo Sakaguchi,4
and Koichi Akashi1,5,*
1Department of Cancer Immunology and AIDS
Dana-Farber Cancer Institute and
2Department of Genetics and
The Howard Hughes Medical Institute
Harvard Medical School
Boston, Massachusetts 02115
3Department of Hematology/Oncology
Boston, Massachusetts 02115
4Department of Immunology
Graduate School of Medical Sciences
1-1-1 Honjo, Kumamoto 860-8556
5Center for Cellular and Molecular Medicine
Kyushu University Hospital
3-1-1 Maidashi, Higashiku, Fukuoka 812-8582
The developmental origin of type I interferon (IFN)-
producing plasmacytoid dendritic cells (PDCs) is con-
globulin heavy chain (IgH) genes in murine PDCs and
the expression of pre-T cell receptor ? (pT?) gene
by human PDCs were proposed as evidence for their
“lymphoid” origin. Here we demonstrate that PDCs
capable of IFN production develop efficiently from
both myeloid- and lymphoid-committed progenitors.
found in both myeloid- and lymphoid-derived PDCs.
The human pT? transgenic reporter was activated in
both myeloid- and lymphoid-derived PDCs at a level
comparable to pre-T cells. PDCs were the only cell
population that activated murine RAG1 knockin and
human pT? transgenic reporters outside the lymphoid
lineage. These results highlight a unique develop-
mental program of PDCs that distinguishes them from
other cell types including conventional dendritic cells.
Dendritic cells (DCs) are one of the most important cell
types inthe immune systemdue to their uniqueability to
orchestrate an acquired immune response (Banchereau
and Steinman, 1998). Murine DCs have been classified
into two populations, myeloid and lymphoid, on the basis
6These authors contributed equally to this work.
New York, New York 10032.
and plays a critical role in the efficient generation of
classical lineage markers of both T and B cells, which
was proposed to reflect their lymphoid past (Corcoran
et al., 2003). On the other hand, human PDCs can be
generated at least from myeloid progenitors expressing
receptors for macrophage colony-stimulating factor
(M-CSF) (Olweus et al., 1996). Mice deficient in ICSBP,
a critical transcription factor for monocyte development
(Tamura et al., 2000), display the loss of PDCs and
CD8??DCs (Aliberti et al., 2003; Schiavoni et al., 2002),
which can be restored by enforced expression of ICSBP
(Tsujimura et al., 2003). A recent report showed that
lymphoid progenitors give rise to cells of B220?CD11clo
PDC phenotype more efficiently than myeloid progeni-
tors (D’Amico and Wu, 2003). In this study, however, the
function of the resulting cells was not evaluated. It is
thus still unclear whether the lymphoid characteristics
of PDCs reflect their lymphoid origin.
or lymphoid-restricted potentials including CMPs and
CLPs (Akashi et al., 2000; Kondo et al., 1997). In the
current study, we analyzed directly the PDC potentials
of these progenitor populations. Our data show that
lymphoid-restricted CLPs and pro-T cells as well as
myeloid-restricted CMPs and GMPs can generate
IFN-producing PDCs with IgH D-J rearrangement. Fur-
thermore, recombination activation gene-1 (RAG1) tran-
scripts were found in both myeloid and lymphoid PDCs.
Themajority ofPDCs includingCMP-derived PDCsacti-
vated RAG1 locus in mice carrying a knockin reporter
for murine RAG1. Furthermore, in transgenic mice car-
rying a human pT? reporter construct, human pT? was
or myeloid origin. Taken together, our data demonstrate
that IFN-producing PDCs can originate from both
lymphoid and myeloid pathways and that the lymphoid
characteristics of PDCs represent a unique “ectopic”
activation of the lymphoid programs. PDC-specific acti-
vation of the human pT? reporter in murine hematopoie-
are faithful counterparts of human PDC subsets.
number of PDC progeny was obtained on day 15 in both
cases. In both CMP- and CLP-injected mice, CD11c?
donor-derived progeny (CD45.2?) in the spleen con-
tained a significant fraction of B220?PDCs as well as
B220?DCs (Figure 1A). PDC and DC development was
observed also in mice injected with 1 ? 105GMPs or
1 ? 104pro-T cells, whereas 1 ? 105MEPs or pro-B
cells did not give rise to detectable numbers of PDC or
DC subsets (Figure 1A and data not shown). Thus, PDC
development appears similar to conventional DC devel-
opment (Manz et al., 2001; Traver et al., 2000; Wu et al.,
2001) in that they can be efficiently derived from both
granulocyte/monocyte and early T lymphoid pathways.
A recent study showed that a circulating population
expressing CD11c and B220 but not MHCII can give rise
to PDCs, CD8??DCs, and CD8??DCs (del Hoyo et al.,
CD11b were also found in the blood of mice injected
with CMPs or CLPs (Figure 1A, right). These CMP- or
CLP-derived CD11c?I-Ab?DC precursors gave rise to
B220?CD11c?PDCs in the culture with Flt-3L (Brawand
et al., 2002) for 2 days (data not shown), suggesting that
at least a fraction of PDCs can originate from CMPs and
CLPs via the common DC precursor stage.
Figure 1B shows phenotypic characteristics of CMP-
or CLP-derived PDCs and DCs. Within the CD11c?DC
fraction in mice reconstituted with CMPs, Gr-1 expres-
sionwas onlyfoundin B220?PDCs. CMP-derivedPDCs
expressed only negative to low levels of CD11b and DC
CD86. A majority of PDCs expressed CD4 and CD8?. In
both CMP- and CLP-derived PDCs, the expression level
of I-Abwas relatively low as compared to B220?DCs.
PDCs were identical to those of B220?CD11c?PDCs
isolated from the normal spleen (Figure 1B, bottom).
produced significant amount of IFN-? upon exposure
to herpes simplex virus (HSV) in vitro, indicating that
they are functional PDCs (Figure 2A).
On a per cell basis, transplanted CMPs and CLPs
were more efficient than GMPs and pro-T cells for both
PDC and DCproduction (Table 1). CMPsand CLPs gave
rise to 2- to 3-fold higher numbers of conventional DCs
as compared to PDCs. Total mouse bone marrow cells
contains ?10-fold more CMPs than CLPs. Accordingly,
the estimated contribution of myeloid progenitors
and ?30% in the spleen and the thymus, respectively
PDC Potentials of Murine Progenitors Correlate
with Their Conventional DC Potentials
We estimated the relative contribution of myeloid and
lymphoid pathways to PDC development by in vivo re-
constitution assays. Myeloid- and lymphoid-committed
progenitors including CMPs, GMPs, and CLPs (CD45.2)
were purified and transplanted into C57B6-CD45.1 mice
poietic stem cells (HSCs) (Spangrude et al., 1988) to
provide radioprotection. Myeloid or lymphoid differenti-
ation potentials of these lineage-restricted progenitors
have been described, previously (Akashi et al., 2000;
Kondo et al., 1997; Na Nakorn et al., 2002). We injected
2 ? 104CLPs or CMPs, and analyzed their PDC progeny
on day 10, 15, and 20 on FACS by using the phenotypic
definition of Lineage (Lin: CD19, CD3, NK1.1, TCR?, and
sIgM) -negative, B220?, and CD11c?(Asselin-Paturel et
al., 2001; Bjorck, 2001; Nakano et al., 2001). The highest
Comparison of Lineage-Specific Gene Expression
in Myeloid and Lymphoid PDCs
We next tested the myeloid or lymphoid characteristics
of CMP- and CLP-derived PDCs and DCs by evaluating
lineage-related gene expression by semiquantitative
RT-PCR (Akashi et al., 2000). As shown in Figure 2B, all
PDC and DC populations expressed Flt-3 and myeloid
cytokine receptors such as IL-3R? and GM-CSFR? at
similar levels. PDCs expressed higher levels of IL-7R?,
an essential cytokine receptor for T and B cell develop-
ment (Akashi et al., 1997; Peschon et al., 1994), regard-
less of their myeloid or lymphoid origin. Both lymphoid-
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