Content uploaded by Yanfeng Wang
Author content
All content in this area was uploaded by Yanfeng Wang
Content may be subject to copyright.
doi:10.1182/blood-2007-10-117531
Prepublished online January 24, 2008;
Andrey S Shaw, Tony Petrella, Harald Stein, Peter G Isaacson, Fabio Facchetti and David Y Mason
Michael Dictor, Martin-Leo Hansmann, Stefano A Pileri, Martin J Dyer, Silvano Sozzani, Ivan Dikic,
Teresa Marafioti, Jennifer C Paterson, Erica Ballabio, Kaaren K Reichard, Sara Tedoldi, Kevin Hollowood,
cells
Novel markers of normal and neoplastic human plasmacytoid dendritic
http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests
Information about reproducing this article in parts or in its entirety may be found online at:
http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints
Information about ordering reprints may be found online at:
http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml
Information about subscriptions and ASH membership may be found online at:
digital object identifier (DOIs) and date of initial publication.
theindexed by PubMed from initial publication. Citations to Advance online articles must include
final publication). Advance online articles are citable and establish publication priority; they are
appeared in the paper journal (edited, typeset versions may be posted when available prior to
Advance online articles have been peer reviewed and accepted for publication but have not yet
Copyright 2011 by The American Society of Hematology; all rights reserved.
20036.
the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
1
Novel markers of normal and neoplastic human plasmacytoid dendritic cells
Running Head: Immunoprofile of normal and neoplastic pDC
Teresa Marafioti
1
, Jennifer C. Paterson
1,*
, Erica Ballabio
1,*
, Kaaren K. Reichard
2,*
,
Sara Tedoldi
1
, Kevin Hollowood
3
, Michael Dictor
4
, Martin-Leo Hansmann
5
, Stefano A.
Pileri
6
, Martin J. Dyer
7
, Silvano Sozzani
8
, Ivan Dikic
9
, Andrey S. Shaw
10
, Tony
Petrella
11
, Harald Stein
12
, Peter G. Isaacson
13
, Fabio Facchetti
8
, and David Y.
Mason
1
1
Leukaemia Research Fund Immunodiagnostics Unit, Nuffield Department of Clinical
Laboratory Sciences, John Radcliffe Hospital, Oxford, United Kingdom;
2
Department
of Pathology, University of New Mexico, Albuquerque, NM;
3
Department of Cellular
Pathology, John Radcliffe Hospital, Oxford, United Kingdom;
4
Department of
Pathology, Lund University Hospital, Lund, Sweden;
5
Senckenberg Pathology
Institute, Johann Wolfgang Goethe-University Clinic Frankfurt am Main, Germany;
6
Haematopathology Unit, "L&A Seragnoli" Institute of Haematology, University of
Bologna, Italy;
7
MRC Toxicology Unit, University of Leicester, United Kingdom;
8
Department of Pathology and General Pathology, University of Brescia, Italy;
9
Institute of Biochemistry II, Goethe University School of Medicine, University Clinic
Frankfurt am Main, Germany;
10
Department of Pathology, Washington University
School of Medicine, St. Louis, MO;
11
Department of Pathology and Centre of
Pathology, Hospital-University Centre, Dijon, France;
12
Institute for Pathology,
Campus Benjamin Franklin, Charité University Medicine Berlin, Germany;
13
Department of Pathology, University College London, London, United Kingdom
Corresponding author’s address: Dr. Teresa Marafioti, Leukaemia Research Fund
Immunodiagnostics Unit, Nuffield Department of Clinical Laboratory Sciences, John
Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom.
* J.C.P., E.B. and K.R. contributed equally to this work.
The work was carried out in the Leukaemia Research Fund Immunodiagnostics Unit,
Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, Oxford,
United Kingdom.
Blood First Edition Paper, prepublished online January 24, 2008; DOI 10.1182/blood-2007-10-117531
Copyright © 2008 American Society of Hematology
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
2
Abstract
Plasmacytoid dendritic cells (pDC) are involved in innate immunity (eg by secreting
interferons) and also give rise to "CD4+CD56+ hematodermic neoplasms". We report
extensive characterization of human pDC in routine tissue samples, documenting the
expression of 19 immunohistologic markers, including signaling molecules (eg
BLNK), transcription factors (eg ICSBP/IRF8 and PU.1) and Toll-like receptors (TLR7
and TLR9). Many of these molecules are expressed in other cell types (principally B
cells), but the adaptor protein CD2AP was essentially restricted to pDC, and is
therefore a novel immunohistologic marker for use in tissue biopsies. We found little
evidence for activation-associated morphologic or phenotypic changes in conditions
where pDC are greatly increased (eg Kikuchi’s disease). Most of the molecules were
retained in the majority of pDC neoplasms, and three (BCL11A, CD2AP and
ICSBP/IRF8) were also commonly negative in "leukemia cutis" (acute myeloid
leukemia in the skin), a tumor that may mimic pDC neoplasia. In summary, we have
documented a range of molecules (notably those associated with B cells) expressed
by pDC in tissues and peripheral blood (where pDC were detectable in cytospins at a
frequency of <1% of mononuclear cells) and also defined potential new markers (in
particular CD2AP) for the diagnosis of pDC tumors.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
3
Introduction
Hematologists are familiar with the major cell lineages generated in the bone marrow
and with the neoplastic disorders to which they give rise. However, less attention has
been focussed on minor hematopoietic subpopulations that, despite their low
numbers have important functional roles and are of clinical relevance because of
their capacity to undergo neoplastic transformation.
One such lineage comprises the cell type known as “plasmacytoid dendritic
cells” (pDC), a population that is typically found in cell clusters (or as isolated cells) in
T cell-rich interfollicular areas in peripheral lymphoid tissue.
1,2
Their “plasmacytoid”
morphology reflects a rich content of rough endoplasmic reticulum whose major
product comprises type I interferons (mainly interferon-
α
). They respond to a variety
of stimuli (including viruses, and hypo- and non-methylated bacterial DNA
sequences
3-6
) and carry receptors, including a number of Toll-like receptors, capable
of binding a spectrum of pathogen-associated molecules.
7
They therefore represent a
strategically positioned first line defence against viral and other pathogens entering
lymphoid tissue from the circulation.
A number of studies have documented the molecules expressed at the RNA
and/or protein level by pDC, although more data are available for the mouse than for
man.
8
Knowledge of the phenotype of pDC is not only of relevance to an
understanding of their origin and function but is also of potential clinical importance in
the field of hemato-oncology since it has been recognized for a number of years that
pDC can undergo neoplastic transformation. In early reports it was noted that such
neoplasms were associated with a myeloproliferative disorder (principally acute or
chronic myelomonocytic or monocytic leukemia),
9
but more recently a distinctive
tumor type, “CD4+C56+ hematodermic neoplasm” involving both the skin and
peripheral blood/bone marrow, has been documented.
10
The diagnosis of these tumors in biopsy samples is based principally on
markers such as CD4, CD56, CD123 and TCL1,
10-12
but most of these are present on
other cell types. Furthermore, difficulties in diagnosis can also arise when a molecule
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
4
is absent or is aberrantly expressed. There is therefore a need for robust additional
markers of pDC detectable in routine biopsies that are expressed on their neoplastic
counterparts but are not found on tumors with which they may be confused.
In this paper we report an extensive immunophenotypic characterization of
human pDC and document a range of molecules (eg involved in cell signaling and
gene transcription) that have not previously been demonstrated in pDC in routine
biopsy material. Many of these are also expressed on neoplastic pDC, and one
molecule, the adaptor protein CD2AP, is not expressed to a comparable degree by
other peripheral white cells, making it a valuable new marker for detecting normal
and neoplastic pDC in tissue biopsies and in peripheral blood.
Materials and methods
Tissue samples
Paraffin-embedded tissue sections of reactive human tonsils, lymph nodes with
histologic features of Castleman’s disease (no. 4), of Kikuchi’s disease (no. 4), skin
biopsies of cutaneous lupus erythematosus (no. 6), lichen planus (no. 5) and normal
bone marrow trephines (no. 2) were obtained from the authors’ institutions. Cryostat
sections of human tonsils from the same source were also used for this study.
Tissue sections from 47 pDC-derived neoplasms were obtained from the
author’s institutions (M.D., F.F., P.G.I., P.T., T.M., K.R. and H.S.) and comprised:
a) Cutaneous, bone marrow or splenic tumors, which had been diagnosed as "blastic
NK-cell lymphoma",
13
"CD4+CD56+ hematodermic neoplasm"
10
or simply (in the
case of two cutaneous neoplasms) as "pDC tumors" (41 cases).
b) Neoplasms diagnosed as "pDC proliferations associated with myeloproliferative
disorders"
9
(6 cases).
Table 1 summarises the available phenotype and clinical data for each case of
pDC neoplasia. The study also included tissue sections of: a) 24 acute myeloid
leukemia in the skin (“leukemia cutis”), with and without CD56 expression, b) bone
marrow trephines from seven chronic myeloid and five chronic myelomonocytic
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
5
leukemias, and c) tissue sections from B- and T-lymphoblastic leukemia/lymphomas
(seven and six cases respectively). This material was retrieved from the files of six
authors (F.F., K.H., M.-L.H., T.M., D.Y.M. and S.P.). The diagnosis of all lymphoid
and myeloid neoplasms was based on the criteria of the WHO classification.
13
Approval from the Oxford Research Ethics Committee B was obtained for this study
(Ref: C02.162).
Peripheral blood samples
Peripheral blood mononuclear cells (PBMCs) were isolated from human EDTA-
anticoagulated peripheral blood from healthy donors after informed consent by a
conventional gradient centrifugation technique using Histopaque (Sigma-Aldrich,
Gillingham, UK). The isolated PBMCs at 1.25x10
6
/ml were used to prepare cytospins
according to a protocol described elsewhere.
14
PDC purification
Peripheral blood mononuclear cells were isolated from buffy coats by Ficoll gradient
(Amersham Biosciences) and pDC were magnetically sorted with blood DC Ag
BDCA-4 cell isolation kits (Miltenyi Biotec), as described previously.
15
More than 90%
of the purified cells expressed CD123 (assessed by flow cytometry – data not
shown), confirming the plasmacytoid DC nature of the great majority of the isolated
cells. Blood pDC isolated in this way (10
6
cells/ml) were cultured in medium
containing 1,000 U/ml recombinant human GM-CSF (Myelogen; Schering-Plough,
Dardilly, France) and 20 ng/ml IL-3 (ProSpec). PDC were finally collected as cytospin
and fixed in 95% ethanol for 5 minutes before immunostaining.
Antibodies
The antibodies used in this study for immunohistologic staining are detailed in Table
2.
Immunostaining
Single immunostaining, double immunoenzymatic and immunofluorescent labeling
was performed on tissue sections and on cytospin preparations of peripheral blood
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
6
mononuclear cells, as described previously.
14,16
In some experiments cytospin
preparations were subjected to “antigen retrieval” in a microwave oven in EDTA-
buffer prior to immunostaining.
Results
Detection of novel markers associated with normal pDC
Paraffin-embedded tissue sections of reactive human tonsils containing abundant
pDC were screened with antibodies against a range of leucocyte-associated
molecules in order to identify markers strongly and selectively expressed in these
cells. Some of the molecules evaluated were chosen because they had been
documented in pDC in the literature (but usually only at the level of mRNA
expression, often in mice) and none had been studied previously in human tissue by
immunohistologic techniques (with the exception of BCL11A, reported recently in
pDC in a single publication
17
). The other molecules evaluated were randomly
selected known leucocyte-associated markers.
The adaptor protein CD2AP emerged from this immunohistologic screening as
a selective marker of pDC, being expressed uniformly throughout the cytoplasm of
these cells (Figure 1). It was not found in other cells in peripheral lymphoid tissue
(with the exception of very weak labeling of mantle zone B cells in some samples and
rare cells in germinal centers). CD2AP was originally cloned from T cells,
18-20
but in
the present study peripheral T cells were consistently CD2AP negative (using three
different antibodies) regardless of antibody dilution or whether the tissue section
came from a paraffin embedded or a cryostat sample. Endothelial cells and tonsillar
squamous epithelium were weakly to moderately positive. In addition, 18 other
molecules were expressed by cells with the typical features of pDC, as summarized
in Table 3. These markers differed greatly in the degree to which they were
expressed in cell types other than pDC but many molecules were also present in B
cells (see Table 3).
Double immunolabeling was performed to explore the relationship between the
new cytoplasmic pDC marker CD2AP and the known pDC-associated transcription
factor BCL11A. This revealed that BCL11A and CD2AP were expressed in the same
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
7
cells (Figure 1), and in double immunoenzymatic labeling studies of the phenotype of
pDC (see Figures 1-4) these two markers were therefore used interchangeably to
reveal pDC (eg CD2AP was used in combination with nuclear markers and BCL11A
in combination with cytoplasmic molecules).
A number of B cell-associated transcription factors, namely ICSBP/IRF8, the
products of the E2A gene (E12 and E47) and FOXP1 (Figure 1) were found in pDC,
and PU.1 was very weakly expressed in a minority of these cells. In contrast, eleven
other transcription factors (eg BCL-6, BOB.1 and PAX-5 - see Table 4), all but three
of which are associated with the B cell lineage, were absent (Figure 1). The
expression of leucocyte-associated molecules involved in intracellular signaling was
also explored and this revealed the presence of five B cell-associated molecules (the
adaptor protein BLNK, the kinases BTK, Lyn and Syk, and the PLC
γ
2 phospholipase)
and also the transmembrane adaptor protein LIME (which is found in T cells and
plasma cells) (Figure 2, Table 3). However, other T cell-associated signaling
molecules (eg TRIM, SLP76) were absent (Figure 2, Table 4). In addition, three
signaling molecules that have not been studied previously in human tissues by
immunocytochemistry were detected in pDC, namely DAP12, IRAK1 and TCB1D4
(Figure 2, Table 3). Furthermore, the Toll-like receptors TLR7 and TLR9 were clearly
present in human pDC (Figure 3, Table 3), in keeping with data from studies of
murine cells.
21,22
Two other cell types present in interfollicular areas in human tonsil, namely
classical interdigitating dendritic cells (expressing DC-SIGN) and presumptive
immature lymphoid cells (expressing TdT), were investigated by double labeling
(immunofluorescence for DC-SIGN and immunoenzymatic for TdT) in combination
with CD2AP (Figure 3). There was no overlap in expression of these markers,
providing further evidence that CD2AP is confined to pDC.
pDC have been extensively evaluated in the past for classical surface/"CD"
molecules associated with lymphoid and myeloid lineages and they are known to
express only a small number, eg CD4, CD68. Such lineage markers were therefore
not explored in detail in this study but we confirmed the expression of CD4 and CD68
in CD2AP-positive cells and also noted that CD79b (but not its partner CD79a) was
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
8
weakly expressed. Furthermore, we studied CD123 (a well recognized marker of
pDC) in relation to the transcription factor BCL11A by double labeling and showed
the latter molecule in all CD123-positive cells (Figure 3) further confirming its
selectivity for pDC. CD33, which has been reported in some pDC-derived
neoplasms,
23,24
was also studied and it was found on occasional pDC (as defined by
expression of BCL11A).
pDC in biopsies from reactive conditions
The pDC that are known to accumulate in non-neoplastic conditions (Kikuchi's
lymphadenitis,
25,26
Castleman’s disease,
27,28
lupus erythematosus
29,30
and lichen
planus
31
) were all sharply delineated (mainly in the form of nodular aggregates but
also as scattered cells) by immunostaining for CD2AP (Figure 3). In biopsies of
cutaneous lupus lesions the pDC appeared identical to those seen in the tonsil,
whereas some of the pDC in Kikuchi's disease were a little larger, with more
voluminous cytoplasm. The phenotypic profile of pDC in the latter disease was
investigated with the full panel of markers to see if it differed from that of tonsillar
pDC, but no differences were observed (Figure 3). It was also noted that CD2AP-
positive cells in Kikuchi’s lymphadenitis were myeloperoxidase-negative (observed
by using double immunofluorescence), showing they were not immature monocytes
(usually present in this disease and positive for myeloperoxidase).
pDC in blood and bone marrow
Cytospin preparations of normal peripheral blood mononuclear cells contained rare
CD2AP-positive cells, accounting for less than 0.5% of all cells (in two healthy
donors) (Figure 4). Their morphology was relatively constant, ie medium-sized cells,
often with slightly eccentrically placed nuclei some of which were indented. These
CD2AP-positive cells all co-expressed the pDC-associated transcription factor
BCL11A and they lacked CD3 and CD20 (Figure 4). Furthermore, pDC isolated from
peripheral blood using anti-BDCA-4 all showed strong staining for CD2AP (Figure 4)
and CD123. BDCA-4 is a well-accepted marker for purification of pDC,
32,33
and it was
used previously by two authors of this paper (FF and SS) to purify pDC, achieving a
purity of 90-98%.
34
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
9
Bone marrow trephine biopsies also contained scattered CD2AP-positive cells,
very similar in morphology to those seen in peripheral lymphoid tissue, and all co-
expressed the transcription factor BCL11A (Figure 4).
pDC neoplasms
A total of 47 pDC-derived neoplasms (detailed in Table 1) were investigated for the
expression of the pDC-associated molecules listed in Table 3 (with the exception of
IRF7, LIME and PLCγ2). However, because of the limited material available, it was
not possible to test each case with all markers. In addition, biopsies from cases of
acute myeloid leukemia in the skin ("leukemia cutis") were studied, since these can
cause diagnostic confusion, together with samples of chronic myeloproliferative
disorders and acute lymphoblastic leukemia (see Table 5).
Most of the pDC markers we evaluated were expressed on the great majority of
cases of pDC neoplasia (Figure 5, Table 5). One of the 16 pDC-associated
molecules, CD2AP, appeared to have particular potential diagnostic value since it
was present in neoplastic pDC in the great majority of cases (41/43), but absent in all
but one of the 24 "leukemia cutis" cases analyzed (and in all other myeloid
neoplasms studied). The B cell-associated transcription factors BCL11A and
ICSBP/IRF8 were also commonly found in neoplastic pDC (42/44 and 34/36 cases
respectively), but they were also found in a minority of cases of "leukemia cutis" (six
and five out of 24 cases respectively). Many of the other pDC-associated molecules
studied (eg BTK, DAP12, Lyn) were found in all cases of pDC neoplasia, but were
also expressed in the majority of “leukemia cutis” samples. Furthermore, markers
expressed in normal B cells (eg BLNK, BTK, Lyn) were commonly found in B cell
acute lymphoblastic leukemia (Table 5).
Discussion
Plasmacytoid dendritic cells were first identified fifty years ago by Lennert and his
associates in human lymph nodes as a population of cells with morphologic features
of plasma cells lying in interfollicular T cell-rich areas.
35
It was subsequently shown
that they express CD36 and CD68, suggesting a monocyte/macrophage origin and
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
10
they came to be known by many authors as "plasmacytoid monocytes".
36
This was
supported by reports that neoplastic proliferations involving these cells (eg in lymph
node, spleen, bone marrow) are always associated with a myeloproliferative disorder
(principally acute or chronic myelomonocytic or monocytic leukemia).
37-41
This
suggested that both proliferative processes share a common origin, and this has
been supported more recently by reports of identical cytogenetic abnormalities in the
two cell populations in cases of myelodysplasia,
42,43
and in a case of acute myeloid
leukemia.
44
"Plasmacytoid monocytes/T cells" were studied by many pathologists in the
following years, and prominent clusters of these cells were recognized as a feature of
Kikuchi's disease,
25,26
and the hyaline vascular subtype of Castleman's disease.
27,28
However, they received relatively little attention until it was shown that they secrete
large amounts of type I interferons (principally interferon-
α
),
1,45
a function that
accounts for their extensive rough endoplasmic reticulum.
29
It was also reported that
similar cells could be generated in vitro from mononuclear or CD34-positive cells in
the peripheral blood and that activation by factors such as IL3 or CD40L generates
cells of dendritic morphology with antigen-presenting capability.
4,46,47
For this reason
they have come to be widely known as "plasmacytoid dendritic cells".
3,4
At the same
time a murine counterpart was identified with similar properties,
48-51
and many
published studies are based on pDC from this species.
It has been reported in recent years that pDC can give rise to a second tumor
type that appears distinct from the rare neoplasms associated with myeloproliferative
disorders referred to above.
23,52-58
This tumor entity was initially believed to arise from
natural killer (NK) cells (because of its expression of CD56
59-61
and categorized as
“blastic NK cell lymphoma” in the WHO classification, but it was subsequently re-
named "CD4+/CD56+ hematodermic neoplasm" in the WHO/EORTC classification
scheme for cutaneous lymphomas.
10,62
These tumors have many phenotypic features
of pDC,
54,63,64
and are characterized by skin lesions, frequent involvement of other
tissues (marrow, spleen and/or lymph nodes), either initially or later in the course of
the disease, and circulating neoplastic cells. The disease often responds initially to
therapy but overall it has a poor prognosis.
23,55-57,61,63-67
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
11
In leukemic cases, the diagnosis is made principally by flow cytometry.
66,68
However, cases are commonly first diagnosed in a skin biopsy, requiring markers
that can be detected in paraffin-embedded tissue.
63
The first markers to be used
(CD4, CD56, HLA-DR, CD123 and CD45RA
54,63
) have more recently been
supplemented by TCL1 and CLA (HECA-452),
10-12,57
but all these markers are also
present on neoplasms of non-pDC origin. A type II C-type lectin BDCA-2 has also
been reported as a specific marker of pDC,
69
and evaluated on pDC tumors.
64,70-72
However, there is little information on its expression in non-pDC neoplasms and it is
only expressed in a proportion of pDC tumors. Siglec-H has also been reported as a
surface constituent on pDC,
73,74
but not exploited as a diagnostic marker.
In this paper we report a number of new immunohistologic markers that can be
used to detect normal and neoplastic pDC in human tissue samples. The adaptor
protein CD2AP (CD2-associated protein) is of particular interest because it was
essentially restricted to pDC. CD2AP is a 80 kDa molecule first identified through its
binding to the terminal 20 amino acids in the cytoplasmic domain of the T cell-
associated molecule CD2.
18,19
CD2AP has also been shown to play a role in
podocyte homeostasis in the kidney (CD2AP-deficient mice develop nephrotic
syndrome and glomerulosclerosis
75,76
). CD2AP is described in the literature as a
component of normal T cells (and we expected to see expression in these cells when
assessing its immunohistologic reactivity in peripheral lymphoid tissues). However,
the only staining observed in T cells was in cortical thymocytes (data not shown) and
three different antibodies (all of which react with cells transfected with the CD2AP
gene - manuscript in preparation) gave identical immunohistologic labeling.
Furthermore, we confirmed by double staining for BCL11A and CD2 (data not shown)
reports from the literature that CD2 (the partner for CD2AP) is not detectable in pDC
in tissue sections
2
(although it has been reported in a minority of cases of pDC
neoplasia).
2,53,57,63,64,66,77
In consequence, CD2AP may associate with a hitherto
unidentified partner other than CD2 in normal pDC. It may be added that, while
CD2AP expression has been documented in mouse T cells, its expression pattern in
human T cells is unclear having only been demonstrated to be expressed in the T
cell leukemia cell line,
Jurkat cells.
18,19,78,79
It is therefore possible that there are
species-specific differences in the pattern of CD2AP expression. It may be added
that Northern blotting studies have suggested that CD2AP is widely expressed in
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
12
human tissues (with the exception of brain),
18,80
but Dustin et al 1998 reported a
more restricted range of protein expression (assessed by Western blotting).
80
Whatever the explanation of the unexpected absence of immunostaining for CD2AP
in peripheral T cells in human tissue samples, it clearly emerged in this empirical
study as a selective and reproducible marker of pDC.
Nine other signaling molecules were also shown for the first time in this study to
be detectable at the protein level in normal and neoplastic human pDC (see Table 3).
Some had been implicated previously at least as possible components of pDC,
usually in murine studies, ie BLNK, Btk, DAP12 (DNAX-activation protein 12, known
also as KARAP), IRAK1 and Syk. However, others have not been associated with
this cell type, eg the GTPase-activating protein TBC1D4 (also known as AS160).
Also the strong expression of TLR7 and TLR9 we found in human pDC (Figure 3) is
in keeping with reports of their transcripts in human and murine pDC.
22,81
82
There is evidence that pDC can arise from both lymphoid and myeloid
precursors,
83-88
and it has also been reported that they show features associated with
several lineages, ie T cells, B cells and myeloid cells.
58,77,85,89,90
Furthermore, the
lymphoid precursor-associated marker TdT is expressed at the mRNA level in murine
pDC and as protein in neoplastic human pDC.
8,56,63,65,91-93
In consequence, the
cellular origin of pDC has been the subject of controversy, to which the present study
might be expected to bring some new insight. It was striking that many markers
detected in normal and neoplastic pDC are expressed in B lymphoid cells (see Table
3), including four transcription factors (BCL11A, the products of the E2A gene,
ICSBP/IRF8 and PU.1) and five signaling molecules (BLNK, BTK, Lyn, PLCγ2 and
Syk). In addition, LIME and TBC1D4 are found in B cells at the plasma cell
94
and
germinal center maturation stage respectively (Figure 2). This suggestion that pDC
are related to B cells is in keeping with reports that signaling initiated from BDCA-2
(CD303) resembles the effect of B cell receptor ligation (eg both involve Syk and
BLNK phosphorylation).
95,96
Interestingly, the consequence of this stimulation is to
inhibit rather than to stimulate IFN production.
69,97,98
However, five classical B cell-
associated transcription factors we studied (eg OCT2, PAX5) were not expressed by
pDC (Table 4). Furthermore, three other B cell-associated molecules we identified
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
13
(Lyn, PU.1 and Syk) are also found in myeloid cells, and another novel marker,
DAP12, is expressed in macrophages but not in B cells (Figure 2, Table 3).
Thus, the phenotypic studies in this paper shed only limited light on the
controversial question of the cellular origin of pDC. We also found no evidence to
support the report by Pelayo et al
8
that different populations of plasmacytoid dendritic
cells ("pDC1" and "pDC2") can be distinguished on the basis of phenotypic
differences. They reported that BCL11A, ICSBP/IRF8, PAX5 are all positive in pDC1
and negative in pDC2, but in the present study we found that these markers (and
indeed other markers evaluated) were either present (BCL11A, ICSBP/IRF8) or
absent (PAX5, mb1/CD79a) in all pDC. The homogeneity of pDC marker expression
in the present paper therefore argues against the existence of subsets of human
pDC1 comparable to those reported by Pelayo et al in mice. It has also been
suggested that the expression of cell markers (eg BDCA-4 and CD7) changes during
pDC maturation, and that the clinical behaviour of pDC-derived tumors is related to
the maturation stage from which they arise.
71
However, the markers documented in
the present study did not show variability suggestive of major phenotypic alteration
during differentiation.
The other controversial topic concerns the degree to which circulating pDC
become cells with classical dendritic morphology, possessing the ability to present
antigen, when they enter peripheral lymphoid tissue. This is a widely held belief, and
accounts for the use of the term "dendritic" and for the fact that on occasion
circulating pDC are referred to as "precursors", implying that their morphology
changes when they emigrate into tissues. In the present study the morphology of
cells in peripheral lymphoid tissue carrying pDC markers appeared very similar to
that of the cells seen in the bone marrow and in the circulation. Furthermore,
although some of the numerous pDC present in Kikuchi’s disease showed slightly
more voluminous cytoplasm for the most part they lacked dendritic surface processes
(Figure 3) and we detected no acquired novel phenotypic features. It is therefore
possible that the ability of pDC to transform into cells with the morphology and
function of classical dendritic cells is an in vitro phenomenon rather than a common
physiologic event in vivo.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
14
A major aim of the present paper was to identify new markers present on
neoplastic pDC. We therefore analyzed a total of 43 cases of hematodermic
neoplasia and also six cases of pDC proliferation associated with a myeloproliferative
disorder (Tables 1 and 5). The phenotype of these two types of pDC neoplasia
matched published data, including the fact that in the latter disorder TdT was not
expressed and CD56 and TCL1 were less commonly expressed than in
hematodermic neoplasia (Table 1). A significant diagnostic problem is posed by
cutaneous deposits of diseases such myeloid leukemia (“leukemia cutis”) since these
are morphologically similar to hematodermic neoplasms and some express CD4 and
CD56. We therefore evaluated the most selective pDC markers on 24 cases of this
sort.
This analysis showed that many of the markers we documented on normal pDC
were expressed on their neoplastic equivalents. Most of these markers were also
expressed on neoplasms of B cell and/or myeloid lineage. However only a minority of
leukemia cutis samples (the neoplasms most likely to be confused with the
CD4+CD56+ hematodermic neoplasm) expressed BCL11A and BLNK, while CD2AP
and ICSBP/IRF8 were even less commonly expressed (Table 5), indicating their
potential value for the diagnosis of hematodermic neoplasms. It should be added that
the frequency of CD2AP and ICSBP/IRF8 expression on myeloid neoplasms was
comparable to, or lower than, that of the two recently proposed diagnostic markers
TCL1 and HECA452/CLA.
11,12,57
The number of cases associated with a myeloproliferative disorder was small
but it may be of significance that some cases in this category lacked the transcription
factors BCL11A (2/5), E47 (1/3) and ICSBP (2/3), whereas these molecules were
present without exception in cases of hematodermic neoplasia. Conversely, PU.1 (a
transcription factor found not only in B cells but also in macrophages) was present in
each of three cases associated with a myeloproliferative disorders but absent in
11/16 hematodermic tumors.
In conclusion, we have documented a range of phenotypic markers of normal
and neoplastic human pDC in tissues and peripheral blood. Our observations do not
resolve the controversy surrounding the cellular origin of these cells (beyond
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
15
reinforcing reports that they share many molecular features with B cells), but they
present a picture of the plasmacytoid dendritic cell as a cell that changes little in
morphology or phenotype between the bone marrow and peripheral tissue, even
when it accumulates in large numbers in diseases such as Kikuchi’s lymphadenitis,
or following neoplastic transformation. These new markers include molecules (in
particular the adaptor protein CD2AP and the transcription factor ICSBP/IRF8) that
may be of value for the diagnosis of pDC tumors.
Acknowledgments
The authors thank Mr. Ralf Lieberz for his technical assistance, and Mrs. Bridget
Watson for her expertise in the preparation of the manuscript. This work was
supported by Project Grant (No. 0382) and Programme Grant (No. 04061) from the
Leukaemia Research Fund and by the Julian Starmer-Smith Lymphoma Fund. The
authors thank all the members of the French Study Group on Cutaneous Lymphomas
for their collaboration.
Conflict of interest disclosure
The authors confirm that there is no conflict of interest and that there are no
competing financial interests.
Author contribution statement
T.M. designed the project, analyzed and interpreted the data, and wrote the paper.
J.C.P., E.B. and S.T. were responsible for cell and tissue preparations as well as
performing all the immunohistologic experiments. K.K.R., K.H., M.D., M-L.H., S.A.P.
and T.P. provided tissue samples. M.J.D., I.D. and A.S.S. provided novel reagents.
S.S. performed part of the experiments. H.S. and P.G.I. provided tissue samples and
also reviewed the results. F.F. provided tissue samples, reviewed the results and
contributed to writing the paper. D.Y.M. contributed to the design of the study,
interpreted the data and wrote the paper.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
16
Table 1. Clinical and phenotypic data in 47 cases of pDC neoplasms
CD4+CD56+ Hematodermic neoplasm
Case
No.
Age/Sex Site of
involvement
CD4 CD45RA CD43 CD56 CD123 TCL1 TdT
1 77/F Skin + + + + Scat’rd+ nd Scat’rd+
2 73/F Skin + nd nd - nd nd +
3 61/M Skin and
bone marrow
+ + + + (30%) + + -
4 67/M Skin + nd + + + + -
5 60/M Skin + + + + + + +
6 80/M Bone
marrow
+ + + + + + --
7 51/M Bone
marrow
+ + + + + nd --
8 75/M Skin + + + + -- + --
9 78/F Spleen + + + + + + --
10 80/M Skin + + + -- + + --
11 78/M Bone
marrow
+ + + + + + --
12 45/F n.a. + + + + + + --
13 58/M Bone
marrow
+ + + + + + --
14 71/M
Skin + + + + + + +
15 72/M Skin + + + + + nd --
16 30/M Skin + + + +/- + + -/+
17 n.a. n.a. + + + +/- + + +/-
18 31/M Skin + + + + + + -/+
19 57 M Skin, lymph
node and
bone marrow
+ nd + + nd nd +
20 68 F Skin + nd + + nd nd +
21 12 F Skin + nd + + nd nd +
22 25/F Skin + nd + + + + nd
23 77/M Skin +
+
+ + + + -
24 35/F Skin + nd + + + + -
25 80/M Skin + nd
nd
+ nd
nd
nd
26 96/F Skin + nd +
+
+ + + (30%)
27 73/M Skin + nd + + + + -
28 76/M Skin + nd + + + + + (50%)
29 54/F Skin + nd - + + + + (80%)
30 71/M Skin + nd + + + + -
31 63/M Skin + nd nd + + + + (10%)
32 8/M Skin + + + + + + -
33 64/M Lymph node + nd + + nd + +
34 84/M Skin + + + + + + -
35 60/F Skin + nd nd + nd nd nd
36 83/M Skin + nd nd + + + -
37 75/M Skin + nd + + + + -
38 25/M Skin + nd nd + + + -
39 70/F Skin + nd + + nd + -
40 88/F Skin + + + + + + + (10%)
41 72/M Skin + + + + + + -
PDC proliferations associated with a myeloproliferative disorder
42 52/M Lymph node + + + (+) + + -
43 63/F Lymph node + + + (+) + (+) -
44 24/M Lymph node
and skin
+ + + - + (+) -
45 62/F Lymph node + + + - + (+) -
46 65/M Lymph node + nd + (+) + + -
47 71/M Bone
marrow
+ + + - + nd -
+ : All tumor cells are positive; (+) : Almost all tumor cells are weakly positive; +/- : Most of the tumor cells are positive; Scat’rd+ :
Scattered tumor cells are positive; -/+ : Most of the tumor cells are negative; – : All tumor cells are negative; nd : Not done;
n.a. : Data not available
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
17
Table 2
. Antibodies used in present study
Molecule Antibody type
Clone name/
product ref.
Source
BCL11A
17
Mouse monoclonal BCL11A/123c
LRF Monoclonal Antibody Facility, John Radcliffe
Hospital, Oxford, UK.
BCL-6
99
Mouse monoclonal GI191E/A8 Dr Giovanna Roncador, CNIO, Madrid, Spain.
BLIMP-1
100
Mouse monoclonal ROS Dr Giovanna Roncador, CNIO, Madrid, Spain.
BLNK Mouse monoclonal 2B11 Santa Cruz Biotechnology, Santa Cruz, CA.
BTK Rabbit polyclonal Contact the corresponding author for details.
BOB.1
(OCAB-1)
a) Mouse monoclonal
b) Rabbit polyclonal
a) TG14 a) Leica Biosystems, Newcastle upon Tyne, UK.
b) Santa Cruz Biotechnology, Santa Cruz, CA.
CD2 Mouse monoclonal AB75 Leica Biosystems, Newcastle upon Tyne, UK.
CD2AP a) Mouse monoclonal
b) Rabbit polyclonal
Contact the corresponding author for details.
CD3 a) Mouse monoclonal
b) Mouse monoclonal
c) Rabbit polyclonal
a) LN10
b) F7.2.38
a) Leica Biosystems, Newcastle upon Tyne, UK.
b) and c) DAKO, Glostrup, Denmark.
CD4 Mouse monoclonal 4B12 Leica Biosystems, Newcastle upon Tyne, UK.
CD19 Rabbit polyclonal 3574 Cell Signaling Technology, Danvers, MA.
CD20 Mouse monoclonal L26 DAKO, Glostrup, Denmark.
CD25 Mouse monoclonal AC9 Leica Biosystems, Newcastle upon Tyne, UK.
CD33 Mouse monoclonal PWS44 Leica Biosystems, Newcastle upon Tyne, UK.
CD34 Mouse monoclonal QBEND-10 DAKO, Glostrup, Denmark.
CD56 Mouse monoclonal BC56C04 Biocare Medical, Concord, CA.
CD79a
101,102
Mouse monoclonal JCB117,
HM57
LRF Immunodiagnostics Unit, John Radcliffe Hospital,
Oxford, UK.
CD79b
103
Mouse monoclonal B29/123
LRF Immunodiagnostics Unit, John Radcliffe Hospital,
Oxford, UK.
CD123 Mouse monoclonal 7G3 BD Biosciences Pharmingen, San Jose, CA.
DAP12 Goat polyclonal Contact the corresponding author for details.
DC-SIGN Mouse monoclonal DC28 R&D Systems, Abingdon, Oxford, UK.
E2A gene
products
(E12/E47)
a) Mouse monoclonal
(E47)
b) Mouse monoclonal
(E12/47)
a) G127-32
b) G98-271
BD Biosciences Pharmingen, San Jose, CA.
ETS.1 Mouse monoclonal 1G11 Leica Biosystems, Newcastle upon Tyne, UK.
FOXP1
104
Mouse monoclonal JC12 AbD Serotec, Kidlington, Oxford, UK.
FOXP3
105
a) Mouse monoclonal
b) Goat polyclonal
a) 236A/E7
b) EB5294
a) Dr Giovanna Roncador, CNIO, Madrid, Spain.
b) Everest Biotech Ltd, Upper Heyford, UK.
GATA-3 a) Mouse monoclonal
b) Mouse monoclonal
a) HG3-31
b) HG3-35
Santa Cruz Biotechnology, Santa Cruz, CA.
Glycophorin A
Mouse monoclonal JC159 DAKO, Glostrup, Denmark.
Glycophorin C Mouse monoclonal Ret40F DAKO, Glostrup, Denmark.
IRAK1 Mouse monoclonal F-4 Santa Cruz Biotechnology, Santa Cruz, CA.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
18
IRF4/MUM-1
106
Mouse monoclonal MUM1p Prof. Brunangelo Falini, Perugia, Italy.
IRF5 Mouse monoclonal Contact the corresponding author for details.
IRF7 Rabbit polyclonal Contact the corresponding author for details.
IRF8/ICSBP Rabbit polyclonal Contact the corresponding author for details.
LAT
94
Mouse monoclonal LAT01 Prof. Vaclav Horejsí, Prague, Czech Republic.
LIME
94
Mouse monoclonal LIME10 Prof. Vaclav Horejsí, Prague, Czech Republic.
Lyn Mouse monoclonal H-6 Santa Cruz Biotechnology, Santa Cruz, CA.
Myelo-
peroxidase
Rabbit polyclonal A0398 DAKO, Glostrup, Denmark.
NFATc1 Mouse monoclonal 7A6 Santa Cruz Biotechnology, Santa Cruz, CA.
OCT-2 Mouse monoclonal Oct-207 Leica Biosystems, Newcastle upon Tyne, UK.
PAX-5
(BSAP)
a) Mouse monoclonal
b) Mouse monoclonal
a) 24
b) 1EW
a) BD Biosciences, San Jose, CA, USA.
b) Leica Biosystems, Newcastle upon Tyne, UK.
PLC 1 Rabbit polyclonal sc-81 Santa Cruz Biotechnology, Santa Cruz, CA.
PLC 2 Mouse monoclonal B-10 Santa Cruz Biotechnology, Santa Cruz, CA.
PU.1 (Spi.1) Mouse monoclonal G148-74 BD Biosciences, Franklin Lakes, NJ, USA.
SLP-76 Rabbit polyclonal sc-9062 Santa Cruz Biotechnology, Santa Cruz, CA..
Syk Rabbit polyclonal sc-1077 Santa Cruz Biotechnology, Santa Cruz, CA.
TBC1D4/AS1
60
Rabbit polyclonal Contact the corresponding author for details.
T-bet Mouse monoclonal 4B10 Santa Cruz Biotechnology, Santa Cruz, CA.
TdT Mouse monoclonal SEN28 Leica Biosystems, Newcastle upon Tyne, UK.
TLR-7 a) Mouse monoclonal
b) Rabbit polyclonal
Contact the corresponding author for details.
TLR-9 a) Mouse monoclonal
b) Rabbit polyclonal
Contact the corresponding author for details.
TRIM
94
Mouse monoclonal TRIM10 Prof. Vaclav Horejsí, Prague, Czech Republic.
Zap-70 Mouse monoclonal 2F3.2 Millipore, Watford, UK.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
19
Table 3. Novel markers of plasmacytoid dendritic cells
Molecule Nature Labeling in
plasmacytoid
dendritic cells
Expression in
other white
cell types
Comments
Transcription regulators
BCL11A Zinc finger
protein
Nuclear
(stronger than
B cells)
B cells. Detected previously at the RNA and
protein level in murine and human
pDC
8,17
and shown to be overexpressed
by gene expression profiling in human
pDC neoplasms.
107
E47/E12
(E2A
gene
products)
Basic helix-
loop-helix
(bHLH)
protein
Nuclear Most B cells. E47 expression detected at the mRNA
level in murine pDC.
8
Not previously
studied as an immunocytochemical
marker of human pDC.
FOXP1 Forkhead
protein
Nuclear Mantle zone B
cells. Minority
of germinal
center B cells.
Plays a role in transcriptional regulation
of B cell development.
108
Not previously
studied in the context of human pDC.
ICSBP
(IRF8)
Interferon
regulatory
factor
Nuclear Most B cells.
Macrophages.
Detected at the RNA level in murine
pDC.
8,85
ICSBP-deficient mice display
loss of PDCs which can be restored by
enforced ICSBP expression.
109-111
Shown to be overexpressed by gene
expression profiling in human pDC
neoplasms.
107
Not previously studied as
an immunocytochemical marker of
human pDC.
IRF7 Interferon
regulatory
factor
Cytoplasmic Absent. Detected in human pDC at RNA and
protein level (flow cytometry).
112-114
Plays an essential role in mice in
induction of type 1 IFN production upon
virus infection.
115
Stimulation of human
pDC with CpG DNA induces activation
of NF-κB and p38 MAPK via TLR9
signaling and leads to the de novo
expression of IRF7.
116
PU.1
(Spi.1)
Member of
the ETS
family
Weak nuclear
staining in
minority of
cells
Most B cells.
Macrophages.
Detected at mRNA level in murine pDC.
8
The closely related factor Spi.B is
detectable at mRNA level in human and
murine pDC and is required for murine
pDC development.
8,117
Not previously
studied as an immunocytochemical
marker of human pDC.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
20
Signaling molecules
BLNK
(SLP65)
Adapter
protein
Cytoplasmic Most B cells. Phosphorylated in the course of
signaling initiated by CD303 (BDCA-2).
96
Not previously studied in the context of
normal pDC but shown to be
overexpressed by gene expression
profiling in human pDC neoplasms.
107
Btk Kinase Cytoplasmic Most B cells. Has a negative regulatory effect on
murine dendritic cells.
118
BTK-deficient
dendritic cells in humans show an
impaired response to TLR8 signaling.
119
Normal numbers of pDC reported in
children lacking the BTK gene.
120
Not
previously studied as an
immunocytochemical marker in human
pDC.
CD2AP
(CMS)
Adaptor
protein
Cytoplasmic Mantle zone B
cells in some
samples (very
weak).
Not previously studied in the context of
pDC.
DAP12
(KARAP)
Adaptor
protein
Cytoplasmic Germinal
center
macrophages,
scattered cells
in interfollicular
areas.
Involved in signaling in pDC initiated via
TLR9 and Siglec-H.
121-123
Not previously
studied as an immunocytochemical
marker in human pDC.
IRAK1 Kinase Membrane
and
cytoplasmic
Scattered cells
in germinal
center and
interfollicular
areas.
Probable
plasma cells.
Involved in signaling induced by
interleukin-1, Toll-like receptors and
tumor necrosis factor receptor (TNFR)
superfamily molecules linking to the
IRF7 transcription factor.
124
Not
previously studied as an
immunocytochemical marker in human
pDC.
LIME Trans-
membrane
adaptor
molecule
Cytoplasmic Most T cells.
Plasma cells.
94
Not previously studied in the context of
pDC.
Lyn Kinase Cytoplasmic Most B cells.
Myeloid
cells.
125
Plays a role in the generation and
maturation of murine dendritic cells
(pDC not specifically studied).
126
Not
previously studied as an
immunocytochemical marker in human
pDC.
PLC
γ
2
Phospho-
lipase
Weak
cytoplasmic
staining
(compared to
B cells)
Most B
cells.
125
Not previously studied as an
immunocytochemical marker in human
pDC.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
21
Toll-like receptors
TLR7 and
TLR9
Endosome-
associated
Cytoplasmic Some cells in
germinal
centers, in
interfollicular
areas and in
plasma cells.
These intracellular receptors have been
extensively studied as initiators of
signaling in murine pDC.
21,81,131,132
Highly expressed in human pDC.
133
Viral
binding may initially be mediated by
antibodies recognized by Fc receptors
before sensing by TLR7.
134
Detected at
the mRNA level in murine pDC.
8
Not
previously studied as
immunocytochemical markers in human
pDC.
Cell surface molecules
CD79b Associated
with
surface Ig
Cytoplasmic
(weak)
Most B cells,
but mantle
zone cells
stronger than
germinal
center cells.
Detected at the mRNA level in murine
pDC.
8
Not previously studied in the
context of human pDC.
Syk Kinase Cytoplasmic Most B cells.
Myeloid
cells.
125
Cross linking of ILT7/Fc
ε
RI complexes
or CD303 (BDCA-2) on pDC causes
phosphorylation of Syk.
95,96,127
Not
previously studied as an
immunocytochemical marker in human
pDC.
TBC1D4/
AS160
GTPase-
activating
protein
Cytoplasmic
Many cells in
germinal
centers
Key regulator of intracellular vescicular
trafficking.
128
Involved in insulin induced
signaling and phosphorylated by Akt
kinase.
129
High mRNA levels reported in
T cells of patients with atopic
dermatitis.
130
Not previously studied as
an immunocytochemical marker of
human pDC.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
22
Table 4. Transcription factors and signaling molecules not detected in pDC
Molecule Normal expression
Transcription factors
BCL6 Germinal center B cells
BLIMP1 Terminally differentiated B cells
BOB1 B cells
Ets.1 Majority of white cells
FOXP3 Regulatory T cells
GATA3 T cells
IRF5 Subpopulation of B cells
MUM1 Plasma cells
OCT2 B cells
PAX5 B cells
T-BET T cells
Signaling molecules
LAT T cells. Megakaryocytes
NFATc1 Most white cells
PLC
γ
1
T cells
SLP76 T cells. Macrophages
TRIM T cells
Zap70 T cells
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
23
Table 5. Expression of pDC-associated molecules in pDC neoplasms and in myeloid and
lymphoid leukemias.
Transcription factors
pDC
neoplasms
Hemato-
dermic
Myelopro-
liferative
associated
Leukemia
cutis
CML CMML B-ALL T-ALL
BCL11A 39/39
(100%)
3/5
(60%)
6
i
/24
(25%)
1/7
(14%)
3/5
(60%)
7
w
/7
(100%)
4/5
(80%)
E47 16
a
/16
(100%)
2
h
/3
(67%)
20
j
/24
(83%)
5/6
(83%)
5/5
(100%)
7/7
(100%)
6/6
(100%)
FOXP1 6/6
(100%)
2/2
(100%)
7/7
(100%)
1/5
(20%)
1/4
(25%)
n.d. n.d.
ICSBP/IRF8 33
b
/33
(100%)
1/3
(33%)
5
k
/24
l
(21%)
0/5
(0%)
0/5
(0%)
0/6
(0%)
0/6
(0%)
PU.1 5
c
/16
(31%)
3/3
(100%)
19
m
/24
(79%)
5/5
(100%)
5/5
(100%)
2/7
(28%)
0/6
(0%)
Signaling molecules
BLNK 21/21
(100%)
4/4
(100%)
8
n
/24
o
(33%)
0/6
(0%)
0/5
(0%)
7/7
(100%)
1/6
(17%)
BTK 16/16
(100%)
3/3
(100%)
24/24
(100%)
1/6
(17%)
2/4
(50%)
7/7
(100%)
0/6
(0%)
CD2AP 35/37
(95%)
6/6
(100%)
1/24
(4%)
0/7
(0%)
0/5
(0%)
0/7
(0%)
0/5
x
(0%)
DAP12 13/13
(100%)
2/2
(100%)
20/22
p
(91%)
5/5
(100%)
3/4
(75%)
0/7
(0%)
0/5
(0%)
IRAK1 6
d
/6
(100%)
1/1
(100%)
6
q
/7
(86%)
1/5
t
(20%)
3
u
/4
(75%)
n.d. n.d.
Lyn 16/16
(100%)
2/2
(100%)
23
r
/24
(96%)
5/5
(100%)
2/5
v
(40%)
6/7
(86%)
0/6
(0%)
Syk 16
e
/16
(100%)
1/1
(100%)
24/24
(100%)
5/5
(100%)
5/5
(100%)
7/7
(100%)
3/6
(50%)
TCB1D4 2/4
(50%)
1/1
(100%)
3/7
(43%)
0/5
(0%)
0/4
(0%)
n.d. n.d.
Receptor molecules
CD79b 9
f
/15
(60%)
0/2
(0%)
3/24
(12.5%)
0/5
(0%)
0/5
(0%)
6/7
(86%)
1/6
(17%)
TLR7 16/16
(100%)
3/3
(100%)
22/24
(92%)
1/6
(17%)
0/4
(0%)
2/7
(28%)
0/6
(0%)
TLR9 10
g
/14
(71%)
1/2
(50%)
24
s
/24
(100%)
0/5
(0%)
4/4
(100%)
0/7
(0%)
2/5
(40%)
Cases are scored as positive if at least 90% of neoplastic cells were stained. In the footnotes below
“weak and/or weakly positive” indicates a low level of staining by all neoplastic cells.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
24
a
4 out of 16 were weakly positive;
b
2 out of 33 were weakly positive;
c
1 out of 5 was very weak positive;
d
2 out of 6 were weakly positive;
e
2 out of 16 were weakly positive;
f
3 out of 9 were weakly positive;
g
2
out of 10 were very weakly positive;
h
1 out of 2 was weak positive;
i
3 out of 6 were weakly positive;
j
6
out of 20 were weakly positive;
k
2 out of 5 were weakly positive;
l
In 3 out of 24 cases (scored as
negative) a small proportion of atypical cells were positive;
m
1 out of 19 was weak positive;
n
3 out of 8
were very weakly positive;
o
In 2 out of 24 cases (scored as negative) a small proportion of atypical cells
were positive;
p
In 2 out of 22 cases (scored as negative) a small proportion of tumor cells were positive;
q
2 out of 6 cases were weakly positive;
r
1 out of 23 was weak positive;
s
3 out of 24 were weakly
positive;
t
In 1
out of 5 cases (scored as negative) a small proportion of cells was weak positive;
u
All 3
cases were weak positive;
v
In 3 out of 5 cases (scored as negative) 5% to 20% of cells were positive;
w
1 out of 7 was very weak positive;
x
1 out of 5 cases showed focal and weak positivity.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
25
Figure legends
Figure 1:
Immunostaining of pDC in human tonsil. Top row: Left: Clusters of pDC (arrowed and
at higher magnification in the inset) lying close to vessels outside a lymphoid follicle
(Foll.) in the T cell-rich region are strongly stained for the adaptor protein CD2AP.
(Immunoperoxidase technique, hematoxylin counterstain). Right: Double
immunoenzymatic labeling for CD2AP (brown) and the B cell-associated transcription
factor BCL11A (blue) confirms that both molecules are present in the same cells. (No
counterstain). Middle row: Co-expression of three B cell-associated transcription
factors, namely ICSBP (brown), E47 (red) and FOXP1 (brown), in pDC expressing
CD2AP (blue or brown). Note that FOXP1 is also expressed in B cells and in
endothelial cells in a vessel (Vess.). (Double immunoenzymatic staining, hematoxylin
counterstain for E47+CD2AP staining). Third row: The B cell-associated transcription
factors BCL6 (brown) and PAX5 (blue) are expressed in B cell follicles (Foll.), but are
absent from extrafollicular pDC (identified by immunostaining for the cytoplasmic
marker CD2AP in blue or brown). Scattered BCL6- and PAX5-positive B cell nuclei
among the pDC are arrowed. (No counterstain). (Images were acquired on a Nikon
Eclipse E800 microscope [Nikon, Tokyo, Japan] equipped with 10x/0.45 Plan Apo or
20x/0.7, 40x/0.95 and 60x/1.4 Plan Fluor objective lenses [Zeiss]), using a Zeiss
Axiocam digital camera [Zeiss, Oberkochen, Germany], Axiovision 3 image
acquisition software [Zeiss] and Adobe Photoshop 7 image processing and
manipulation software (Adobe, San Jose, CA).
Figure 2:
Immunostaining of human tonsil for signaling molecules in pDC. Top row: BLNK is
clearly present in a cluster of pDC (circled and at higher magnification in the inset)
lying close to a lymphoid follicle (Foll.). (Immunoperoxidase staining, hematoxylin
counterstain). Double immunofluorescent labeling (with CD2AP) shows that pDC also
express the signaling molecules Syk and Btk. (DAPI counterstain for Syk). Middle
row: pDC, identified by double staining for the transcription factor BCL11A (blue),
express the transmembrane adaptor protein LIME (brown) but are negative for two
other T cell-associated signaling molecules (TRIM and SLP76 – both brown). The
arrows indicate LIME-positive BCL11A-negative T cells. (No counterstain). Third row:
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
26
Left: A cluster of pDC (circled and at higher magnification, center) lying adjacent to a
lymphoid follicle (Foll.) co-express DAP12 (brown) and the transcription factor
BCL11A (blue). Macrophages in the follicle (arrowed) express DAP12 alone. (No
counterstain). Right: TCB1B4 (a GTPase activating protein) is also strongly
expressed by clusters of pDC (circled in low power view). Note expression of
TCB1B4 in a B cell follicle (Foll.). (Immunoperoxidase staining, hematoxylin
counterstain). (Images were acquired on a Nikon Eclipse E800 microscope [Nikon,
Tokyo, Japan] equipped with 20x/0.7, 40x/0.95 and 60x/1.4 Plan Fluor objective
lenses [Zeiss]), using a Zeiss Axiocam digital camera [Zeiss, Oberkochen, Germany],
Axiovision 3 image acquisition software [Zeiss] and Adobe Photoshop 7 image
processing and manipulation software (Adobe, San Jose, CA).
Figure 3:
Immunostaining of human tonsil and in disorders characterized by reactive pDC. Top
two rows: pDC express the Toll-like receptors TLR7 and TLR9. (Immunoperoxidase
staining, hematoxylin counterstain). Vess: vessel. Double immunofluorescent labeling
shows co-expression in pDC of TLR7 and CD2AP. Third row: Left: The interfollicular
area contains cells stained for the pDC marker CD2AP (brown) and also TdT-positive
cells (red), but it is evident (see high magnification inset) that these two populations
do not overlap. (Hematoxylin counterstain). Right: Double immunoenzymatic staining
for CD123 (brown) and BCL11A (blue) shows co-expression in pDC. The nuclei in
the background with weak staining for BCL11A are B cells. (No counterstain). Fourth
row: The clusters of pDC that accumulate in cutaneous lupus (revealed by
immunostaining for CD2AP) are morphologically identical to those seen in tonsil and
blood, and show no evidence of dendritic processes. (Immunoperoxidase staining,
hematoxylin counterstain). In Kikuchi’s disease, large clusters of pDC are seen,
some of which have increased cytoplasm, and they show the same pattern of marker
expression as normal pDC, eg positive for the cytoplasmic marker CD2AP (brown or
red), and for the transcription factors BCL11A (red) and ICSBP/IRF8 (brown).
(Immunoperoxidase and double immunoenzymatic staining, hematoxylin
counterstain). (Images were acquired on a Nikon Eclipse E800 microscope [Nikon,
Tokyo, Japan] equipped with 20x/0.7, 40x/0.95 and 60x/1.4 Plan Fluor objective
lenses [Zeiss]), using a Zeiss Axiocam digital camera [Zeiss, Oberkochen, Germany],
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
27
Axiovision 3 image acquisition software [Zeiss] and Adobe Photoshop 7 image
processing and manipulation software (Adobe, San Jose, CA).
Figure 4:
Immunostaining of pDC in blood and bone marrow. Upper panel: Immunoperoxidase
staining of normal peripheral blood mononuclear cells (cytospin preparation) shows
rare cells expressing the pDC marker CD2AP. Double labeling (right) shows that
these cells do not express CD3 or CD20 (double immunofluorescence) but they co-
express (as in tissue) CD2AP (brown) and BCL11A (red) (double staining).
Immunostaining of cytospin preparations of pDC isolated from peripheral blood using
anti-BDCA-4 shows that the great majority of cells express CD2AP and also the pDC
marker CD123 (and no staining is seen in a negative control). CD2AP is also
expressed by pDC isolated from peripheral blood using anti-BDCA-4 (Cytospin
preparation). (DAPI counterstain for the immunofluorescence preparations,
hematoxylin counterstain for the immunoenzymatic preparations). Lower panel:
Scattered pDC (arrowed) are seen in a bone marrow trephine (upper row)
immunostained for CD2AP and glycophorin (in brown and red respectively). In the
lower row, examples are shown at high magnification of bone marrow pDC co-
expressing CD2AP (brown) and BCL11A (red). (Hematoxylin counterstain). (Images
were acquired on a Nikon Eclipse E800 microscope [Nikon, Tokyo, Japan] equipped
with 60x/1.4 and 100/1.3 Plan Fluor objective lenses [Zeiss]), using a Zeiss Axiocam
digital camera [Zeiss, Oberkochen, Germany], Axiovision 3 image acquisition
software [Zeiss] and Adobe Photoshop 7 image processing and manipulation
software (Adobe, San Jose, CA).
Figure 5:
Immunostaining of pDC-associated markers in tumors derived from these cells (left),
and in cutaneous deposits of acute myeloid leukemia ("leukemia cutis") (right), a
neoplasm that can show similar histopathologic features. The markers shown were
expressed by essentially all cases of pDC-derived neoplasms, and the first four were
expressed only in a minority of leukemia cutis biopsies (BCL11A: 6/24 cases; BLNK:
8/24 cases; CD2AP: 1/24 cases and ICSBP: 5/24 cases). In contrast, BTK was
expressed in all samples of leukemia cutis tested (24/24 cases). (Immunoperoxidase
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
28
staining, hematoxylin counterstain). (Images were acquired on a Nikon Eclipse E800
microscope [Nikon, Tokyo, Japan] equipped with 20x/0.7, 40x/0.95 and 60x/1.4 Plan
Fluor objective lenses [Zeiss]), using a Zeiss Axiocam digital camera [Zeiss,
Oberkochen, Germany], Axiovision 3 image acquisition software [Zeiss] and Adobe
Photoshop 7 image processing and manipulation software (Adobe, San Jose, CA).
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
29
References
1. Facchetti F, De Wolf-Peeters C, van den Oord JJ, De vos R, Desmet VJ.
Plasmacytoid T cells: a cell population normally present in the reactive lymph
node. An immunohistochemical and electronmicroscopic study. Hum Pathol.
1988;19:1085-1092.
2. Facchetti F, Vermi W, Mason D, Colonna M. The plasmacytoid
monocyte/interferon producing cells. Virchows Arch. 2003;443:703-717.
3. Grouard G, Rissoan MC, Filgueira L, Durand I, Banchereau J, Liu YJ. The
enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3
and CD40-ligand. J Exp Med. 1997;185:1101-1111.
4. Cella M, Facchetti F, Lanzavecchia A, Colonna M. Plasmacytoid dendritic cells
activated by influenza virus and CD40L drive a potent TH1 polarization. Nat
Immunol. 2000;1:305-310.
5. Bauer M, Redecke V, Ellwart JW, et al. Bacterial CpG-DNA triggers activation
and maturation of human CD11c-, CD123+ dendritic cells. J Immunol.
2001;166:5000-5007.
6. Krug A, Towarowski A, Britsch S, et al. Toll-like receptor expression reveals
CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which
synergizes with CD40 ligand to induce high amounts of IL-12. Eur J Immunol.
2001;31:3026-3037.
7. Kawai T, Akira S. Innate immune recognition of viral infection. Nat Immunol.
2006;7:131-137.
8. Pelayo R, Hirose J, Huang J, et al. Derivation of 2 categories of plasmacytoid
dendritic cells in murine bone marrow. Blood. 2005;105:4407-4415.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
30
9. Vermi W, Facchetti F, Rosati S, et al. Nodal and extranodal tumor-forming
accumulation of plasmacytoid monocytes/interferon-producing cells associated
with myeloid disorders. Am J Surg Pathol. 2004;28:585-595.
10. Willemze R, Jaffe ES, Burg G, et al. WHO-EORTC classification for cutaneous
lymphomas. Blood. 2005;105:3768-3785.
11. Herling M, Teitell MA, Shen RR, Medeiros LJ, Jones D. TCL1 expression in
plasmacytoid dendritic cells (DC2s) and the related CD4+ CD56+ blastic tumors
of skin. Blood. 2003;101:5007-5009.
12. Petrella T, Meijer CJ, Dalac S, et al. TCL1 and CLA expression in agranular
CD4/CD56 hematodermic neoplasms (blastic NK-cell lymphomas) and
leukemia cutis. Am J Clin Pathol. 2004;122:307-313.
13. Jaffe ES, Harris NL, Stein H, Vardiman JW eds. WHO Classification: Tumours
of haematopoietic and lymphoid tissues. In: Jaffe ES, Harris NL, Stein H,
Vardiman JW eds: IARC Press, Lyon (France); 2001.
14. Marafioti T, Pozzobon M, Hansmann ML, Delsol G, Pileri SA, Mason DY.
Expression of intracellular signaling molecules in classical and lymphocyte
predominance Hodgkin disease. Blood. 2004;103:188-193.
15. Penna G, Sozzani S, Adorini L. Cutting edge: selective usage of chemokine
receptors by plasmacytoid dendritic cells. J Immunol. 2001;167:1862-1866.
16. Paterson JC, Ballabio E, Mattsson G, Turner SH, Mason DY, Marafioti T.
Labeling of multiple cell markers and mRNA using automated apparatus. Appl
Immunohistochem Mol Morphol. In press.
17. Pulford K, Banham AH, Lyne L, et al. The BCL11AXL transcription factor: its
distribution in normal and malignant tissues and use as a marker for
plasmacytoid dendritic cells. Leukemia. 2006;20:1439-1441.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
31
18. Dustin ML, Olszowy MW, Holdorf AD, et al. A novel adaptor protein
orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts.
Cell. 1998;94:667-677.
19. Hutchings NJ, Clarkson N, Chalkley R, Barclay AN, Brown MH. Linking the T
cell surface protein CD2 to the actin-capping protein CAPZ via CMS and CIN85.
J Biol Chem. 2003;278:22396-22403.
20. Shaw AS. T-cell activation and immunologic synapse. Immunol Res.
2005;32:247-252.
21. Kaisho T, Akira S. Regulation of dendritic cell function through Toll-like
receptors. Curr Mol Med. 2003;3:373-385.
22. Rothenfusser S, Tuma E, Endres S, Hartmann G. Plasmacytoid dendritic cells:
the key to CpG. Hum Immunol. 2002;63:1111-1119.
23. Jacob MC, Chaperot L, Mossuz P, et al. CD4+ CD56+ lineage negative
malignancies: a new entity developed from malignant early plasmacytoid
dendritic cells. Haematologica. 2003;88:941-955.
24. Garnache-Ottou F, Chaperot L, Biichle S, et al. Expression of the myeloid-
associated marker CD33 is not an exclusive factor for leukemic plasmacytoid
dendritic cells. Blood. 2005;105:1256-1264.
25. Pileri S, Kikuchi M, Helbron D, Lennert K. Histiocytic necrotizing lymphadenitis
without granulocytic infiltration. Virchows Arch A Pathol Anat Histol.
1982;395:257-271.
26. Facchetti F, de Wolf-Peeters C, van den Oord JJ, de Vos R, Desmet VJ.
Plasmacytoid monocytes (so-called plasmacytoid T-cells) in Kikuchi's
lymphadenitis. An immunohistologic study. Am J Clin Pathol. 1989;92:42-50.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
32
27. Harris NL, Bhan AK. "Plasmacytoid T cells" in Castleman's disease.
Immunohistologic phenotype. Am J Surg Pathol. 1987;11:109-113.
28. Danon AD, Krishnan J, Frizzera G. Morpho-immunophenotypic diversity of
Castleman's disease, hyaline-vascular type: with emphasis on a stroma-rich
variant and a new pathogenetic hypothesis. Virchows Arch A Pathol Anat
Histopathol. 1993;423:369-382.
29. Blanco P, Palucka AK, Gill M, Pascual V, Banchereau J. Induction of dendritic
cell differentiation by IFN-alpha in systemic lupus erythematosus. Science.
2001;294:1540-1543.
30. Ronnblom L, Alm GV. A pivotal role for the natural interferon alpha-producing
cells (plasmacytoid dendritic cells) in the pathogenesis of lupus. J Exp Med.
2001;194:F59-63.
31. Santoro A, Majorana A, Roversi L, et al. Recruitment of dendritic cells in oral
lichen planus. J Pathol. 2005;205:426-434.
32. Meyer TP, Zehnter I, Hofmann B, et al. Filter Buffy Coats (FBC): a source of
peripheral blood leukocytes recovered from leukocyte depletion filters. J
Immunol Methods. 2005;307:150-166.
33. Grage-Griebenow E, Loseke S, Kauth M, Gehlhar K, Zawatzky R, Bufe A. Anti-
BDCA-4 (neuropilin-1) antibody can suppress virus-induced IFN-alpha
production of plasmacytoid dendritic cells. Immunol Cell Biol. 2007;85:383-390.
34. Penna G, Vulcano M, Sozzani S, Adorini L. Differential migration behavior and
chemokine production by myeloid and plasmacytoid dendritic cells. Hum
Immunol. 2002;63:1164-1171.
35. Lennert K, Remmele W. [Karyometric research on lymph node cells in man. I.
Germinoblasts, lymphoblasts & lymphocytes.]. Acta Haematol. 1958;19:99-113.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
33
36. Facchetti F, de Wolf-Peeters C, Mason DY, Pulford K, van den Oord JJ,
Desmet VJ. Plasmacytoid T cells. Immunohistochemical evidence for their
monocyte/macrophage origin. Am J Pathol. 1988;133:15-21.
37. Muller-Hermelink HK, Stein H, Steinmann G, Lennert K. Malignant lymphoma of
plasmacytoid T-cells. Morphologic and immunologic studies characterizing a
special type of T-cell. Am J Surg Pathol. 1983;7:849-862.
38. Prasthofer EF, Prchal JT, Grizzle WE, Grossi CE. Plasmacytoid T-cell
lymphoma associated with chronic myeloproliferative disorder. Am J Surg
Pathol. 1985;9:380-387.
39. Beiske K, Langholm R, Godal T, Marton PF. T-zone lymphoma with
predominance of 'plasmacytoid T-cells' associated with myelomonocytic
leukaemia--a distinct clinicopathological entity. J Pathol. 1986;150:247-255.
40. Harris NL, Demirjian Z. Plasmacytoid T-zone cell proliferation in a patient with
chronic myelomonocytic leukemia. Histologic and immunohistologic
characterization. Am J Surg Pathol. 1991;15:87-95.
41. Baddoura FK, Hanson C, Chan WC. Plasmacytoid monocyte proliferation
associated with myeloproliferative disorders. Cancer. 1992;69:1457-1467.
42. Mongkonsritragoon W, Letendre L, Qian J, Li CY. Nodular lesions of monocytic
component in myelodysplastic syndrome. Am J Clin Pathol. 1998;110:154-162.
43. Ferry JA, Harris NL. Plasmacytoid monocytes? Am J Clin Pathol. 1999;111:569.
44. Facchetti F, Vergoni F, Rossi E, et al. Neoplastic nature of nodal plasmacytoid
monocytes associated with acute myeloid leukemia. Mod Pathol. 2002;15:239A.
45. Cella M, Jarrossay D, Facchetti F, et al. Plasmacytoid monocytes migrate to
inflamed lymph nodes and produce large amounts of type I interferon. Nat Med.
1999;5:919-923.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
34
46. Liu YJ. Dendritic cell subsets and lineages, and their functions in innate and
adaptive immunity. Cell. 2001;106:259-262.
47. Shortman K, Liu YJ. Mouse and human dendritic cell subtypes. Nat Rev
Immunol. 2002;2:151-161.
48. Asselin-Paturel C, Boonstra A, Dalod M, et al. Mouse type I IFN-producing cells
are immature APCs with plasmacytoid morphology. Nat Immunol. 2001;2:1144-
1150.
49. Bjorck P. Isolation and characterization of plasmacytoid dendritic cells from Flt3
ligand and granulocyte-macrophage colony-stimulating factor-treated mice.
Blood. 2001;98:3520-3526.
50. Nakano H, Yanagita M, Gunn MD. CD11c(+)B220(+)Gr-1(+) cells in mouse
lymph nodes and spleen display characteristics of plasmacytoid dendritic cells.
J Exp Med. 2001;194:1171-1178.
51. Martin P, Del Hoyo GM, Anjuere F, et al. Characterization of a new
subpopulation of mouse CD8alpha+ B220+ dendritic cells endowed with type 1
interferon production capacity and tolerogenic potential. Blood. 2002;100:383-
390.
52. Bekkenk MW, Jansen PM, Meijer CJ, Willemze R. CD56+ hematological
neoplasms presenting in the skin: a retrospective analysis of 23 new cases and
130 cases from the literature. Ann Oncol. 2004;15:1097-1108.
53. Chaperot L, Bendriss N, Manches O, et al. Identification of a leukemic
counterpart of the plasmacytoid dendritic cells. Blood. 2001;97:3210-3217.
54. Petrella T, Comeau MR, Maynadie M, et al. 'Agranular CD4+ CD56+
hematodermic neoplasm' (blastic NK-cell lymphoma) originates from a
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
35
population of CD56+ precursor cells related to plasmacytoid monocytes. Am J
Surg Pathol. 2002;26:852-862.
55. Feuillard J, Jacob MC, Valensi F, et al. Clinical and biologic features of
CD4(+)CD56(+) malignancies. Blood. 2002;99:1556-1563.
56. Kim Y, Kang MS, Kim CW, Sung R, Ko YH. CD4+CD56+ lineage negative
hematopoietic neoplasm: so called blastic NK cell lymphoma. J Korean Med
Sci. 2005;20:319-324.
57. Petrella T, Bagot M, Willemze R, et al. Blastic NK-cell lymphomas (agranular
CD4+CD56+ hematodermic neoplasms): a review. Am J Clin Pathol.
2005;123:662-675.
58. Garnache-Ottou F, Feuillard J, Saas P. Plasmacytoid dendritic cell
leukaemia/lymphoma: towards a well defined entity? Br J Haematol.
2007;136:539-548.
59. DiGiuseppe JA, Louie DC, Williams JE, et al. Blastic natural killer cell
leukemia/lymphoma: a clinicopathologic study. Am J Surg Pathol.
1997;21:1223-1230.
60. Uchiyama N, Ito K, Kawai K, Sakamoto F, Takaki M, Ito M. CD2-, CD4+, CD56+
agranular natural killer cell lymphoma of the skin. Am J Dermatopathol.
1998;20:513-517.
61. Petrella T, Dalac S, Maynadie M, et al. CD4+ CD56+ cutaneous neoplasms: a
distinct hematological entity? Groupe Francais d'Etude des Lymphomes
Cutanes (GFELC). Am J Surg Pathol. 1999;23:137-146.
62. Chan JKC, Jaffe ES, Ralfkiaer E. Blastic NK-cell lymphoma. In: Jaffe ES, Harris
NL, Stein H, Vardiman JW, eds. WHO Classification: Tumours of
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
36
Haematopoietic and Lymphoid Tissues: IARC Press, Lyon (France); 2001:214-
215.
63. Reichard KK, Burks EJ, Foucar MK, et al. CD4(+) CD56(+) lineage-negative
malignancies are rare tumors of plasmacytoid dendritic cells. Am J Surg Pathol.
2005;29:1274-1283.
64. Urosevic M, Conrad C, Kamarashev J, et al. CD4+CD56+ hematodermic
neoplasms bear a plasmacytoid dendritic cell phenotype. Hum Pathol.
2005;36:1020-1024.
65. Kato N, Yasukawa K, Kimura K, et al. CD2- CD4+ CD56+
hematodermic/hematolymphoid malignancy. J Am Acad Dermatol.
2001;44:231-238.
66. Gopcsa L, Banyai A, Jakab K, et al. Extensive flow cytometric characterization
of plasmacytoid dendritic cell leukemia cells. Eur J Haematol. 2005;75:346-351.
67. Ng AP, Lade S, Rutherford T, McCormack C, Prince HM, Westerman DA.
Primary cutaneous CD4+/CD56+ hematodermic neoplasm (blastic NK-cell
lymphoma): a report of five cases. Haematologica. 2006;91:143-144.
68. Trimoreau F, Donnard M, Turlure P, Gachard N, Bordessoule D, Feuillard J.
The CD4+ CD56+ CD116- CD123+ CD45RA+ CD45RO- profile is specific of
DC2 malignancies. Haematologica. 2003;88:ELT10.
69. Dzionek A, Sohma Y, Nagafune J, et al. BDCA-2, a novel plasmacytoid
dendritic cell-specific type II C-type lectin, mediates antigen capture and is a
potent inhibitor of interferon alpha/beta induction. J Exp Med. 2001;194:1823-
1834.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
37
70. Chaperot L, Perrot I, Jacob MC, et al. Leukemic plasmacytoid dendritic cells
share phenotypic and functional features with their normal counterparts. Eur J
Immunol. 2004;34:418-426.
71. Jaye DL, Geigerman CM, Herling M, Eastburn K, Waller EK, Jones D.
Expression of the plasmacytoid dendritic cell marker BDCA-2 supports a
spectrum of maturation among CD4+ CD56+ hematodermic neoplasms. Mod
Pathol. 2006;19:1555-1562.
72. Pilichowska ME, Fleming MD, Pinkus JL, Pinkus GS. CD4+/CD56+
hematodermic neoplasm ("blastic natural killer cell lymphoma"): neoplastic cells
express the immature dendritic cell marker BDCA-2 and produce interferon. Am
J Clin Pathol. 2007;128:445-453.
73. Blasius AL, Cella M, Maldonado J, Takai T, Colonna M. Siglec-H is an IPC-
specific receptor that modulates type I IFN secretion through DAP12. Blood.
2006;107:2474-2476.
74. Zhang J, Raper A, Sugita N, et al. Characterization of Siglec-H as a novel
endocytic receptor expressed on murine plasmacytoid dendritic cell precursors.
Blood. 2006;107:3600-3608.
75. Shih NY, Li J, Karpitskii V, et al. Congenital nephrotic syndrome in mice lacking
CD2-associated protein. Science. 1999;286:312-315.
76. Huber TB, Hartleben B, Kim J, et al. Nephrin and CD2AP associate with
phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling. Mol Cell
Biol. 2003;23:4917-4928.
77. Comeau MR, Van der Vuurst de Vries AR, Maliszewski CR, Galibert L.
CD123bright plasmacytoid predendritic cells: progenitors undergoing cell fate
conversion? J Immunol. 2002;169:75-83.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
38
78. Harriague J, Debre P, Bismuth G, Hubert P. Priming of CD2-induced p62Dok
tyrosine phosphorylation by CD3 in Jurkat T cells. Eur J Immunol.
2000;30:3319-3328.
79. Badour K, Zhang J, Shi F, et al. The Wiskott-Aldrich syndrome protein acts
downstream of CD2 and the CD2AP and PSTPIP1 adaptors to promote
formation of the immunological synapse. Immunity. 2003;18:141-154.
80. Kirsch KH, Georgescu MM, Ishimaru S, Hanafusa H. CMS: an adapter molecule
involved in cytoskeletal rearrangements. Proc Natl Acad Sci U S A.
1999;96:6211-6216.
81. Ito T, Wang YH, Liu YJ. Plasmacytoid dendritic cell precursors/type I interferon-
producing cells sense viral infection by Toll-like receptor (TLR) 7 and TLR9.
Springer Semin Immunopathol. 2005;26:221-229.
82. Blasius AL, Colonna M. Sampling and signaling in plasmacytoid dendritic cells:
the potential roles of Siglec-H. Trends Immunol. 2006;27:255-260.
83. Olweus J, Thompson PA, Lund-Johansen F. Granulocytic and monocytic
differentiation of CD34hi cells is associated with distinct changes in the
expression of the PU.1-regulated molecules, CD64 and macrophage colony-
stimulating factor receptor. Blood. 1996;88:3741-3754.
84. Chicha L, Jarrossay D, Manz MG. Clonal type I interferon-producing and
dendritic cell precursors are contained in both human lymphoid and myeloid
progenitor populations. J Exp Med. 2004;200:1519-1524.
85. Shigematsu H, Reizis B, Iwasaki H, et al. Plasmacytoid dendritic cells activate
lymphoid-specific genetic programs irrespective of their cellular origin.
Immunity. 2004;21:43-53.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
39
86. Wang YH, Liu YJ. Mysterious origin of plasmacytoid dendritic cell precursors.
Immunity. 2004;21:1-2.
87. Harman BC, Miller JP, Nikbakht N, Gerstein R, Allman D. Mouse plasmacytoid
dendritic cells derive exclusively from estrogen-resistant myeloid progenitors.
Blood. 2006;108:878-885.
88. Ishikawa F, Niiro H, Iino T, et al. The developmental program of human
dendritic cells is operated independently of conventional myeloid and lymphoid
pathways. Blood. 2007;110:3591-3660.
89. Rissoan MC, Duhen T, Bridon JM, et al. Subtractive hybridization reveals the
expression of immunoglobulin-like transcript 7, Eph-B1, granzyme B, and 3
novel transcripts in human plasmacytoid dendritic cells. Blood. 2002;100:3295-
3303.
90. Naik SH, Corcoran LM, Wu L. Development of murine plasmacytoid dendritic
cell subsets. Immunol Cell Biol. 2005;83:563-570.
91. Rakozy CK, Mohamed AN, Vo TD, et al. CD56+/CD4+ lymphomas and
leukemias are morphologically, immunophenotypically, cytogenetically, and
clinically diverse. Am J Clin Pathol. 2001;116:168-176.
92. Khoury JD, Medeiros LJ, Manning JT, Sulak LE, Bueso-Ramos C, Jones D.
CD56(+) TdT(+) blastic natural killer cell tumor of the skin: a primitive systemic
malignancy related to myelomonocytic leukemia. Cancer. 2002;94:2401-2408.
93. Knudsen H, Gronbaek K, thor Straten P, et al. A case of lymphoblastoid natural
killer (NK)-cell lymphoma: association with the NK-cell receptor complex
CD94/NKG2 and TP53 intragenic deletion. Br J Dermatol. 2002;146:148-153.
94. Tedoldi S, Paterson JC, Hansmann ML, et al. Transmembrane adaptor
molecules: a new category of lymphoid-cell markers. Blood. 2006;107:213-221.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
40
95. Cao W, Zhang L, Rosen DB, et al. Plasmacytoid dendritic cell receptor
BDCA2/FcR complex uses BCR-like signaling cascade to modulate Toll-like
receptor responses. J Immunol. 2007;178:89.20.
96. Rock J, Schmitz J, Winkels G. CD303 (BDCA-2) mediated inhibition of
interferon type I production in plasmacytoid dendritic cells is linked to SYK,
BLNK and PKC delta signaling. J Immunol. 2007;178:89.25.
97. Fanning SL, George TC, Feng D, et al. Receptor cross-linking on human
plasmacytoid dendritic cells leads to the regulation of IFN-alpha production. J
Immunol. 2006;177:5829-5839.
98. Cao W, Zhang L, Rosen DB, et al. BDCA2/Fc epsilon RI gamma complex
signals through a novel BCR-like pathway in human plasmacytoid dendritic
cells. PLoS Biol. 2007;5:e248.
99. Garcia JF, Garcia JF, Maestre L, et al. Genetic immunization: a new
monoclonal antibody for the detection of BCL-6 protein in paraffin sections. J
Histochem Cytochem. 2006;54:31-38.
100. Garcia JF, Roncador G, Garcia JF, et al. PRDM1/BLIMP-1 expression in
multiple B and T-cell lymphoma. Haematologica. 2006;91:467-474.
101. Mason DY, Cordell JL, Brown MH, et al. CD79a: a novel marker for B-cell
neoplasms in routinely processed tissue samples. Blood. 1995;86:1453-1459.
102. Mason DY, Cordell JL, Tse AG, et al. The IgM-associated protein mb-1 as a
marker of normal and neoplastic B cells. J Immunol. 1991;147:2474-2482.
103. Mason DY, van Noesel CJ, Cordell JL, et al. The B29 and mb-1 polypeptides
are differentially expressed during human B cell differentiation. Eur J Immunol.
1992;22:2753-2756.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
41
104. Banham AH, Beasley N, Campo E, et al. The FOXP1 winged helix transcription
factor is a novel candidate tumor suppressor gene on chromosome 3p. Cancer
Res. 2001;61:8820-8829.
105. Roncador G, Brown PJ, Maestre L, et al. Analysis of FOXP3 protein expression
in human CD4+CD25+ regulatory T cells at the single-cell level. Eur J Immunol.
2005;35:1681-1691.
106. Falini B, Fizzotti M, Pucciarini A, et al. A monoclonal antibody (MUM1p) detects
expression of the MUM1/IRF4 protein in a subset of germinal center B cells,
plasma cells, and activated T cells. Blood. 2000;95:2084-2092.
107. Dijkman R, van Doorn R, Szuhai K, Willemze R, Vermeer MH, Tensen CP.
Gene-expression profiling and array-based CGH classify CD4+CD56+
hematodermic neoplasm and cutaneous myelomonocytic leukemia as distinct
disease entities. Blood. 2007;109:1720-1727.
108. Hu H, Wang B, Borde M, et al. Foxp1 is an essential transcriptional regulator of
B cell development. Nat Immunol. 2006;7:819-826.
109. Schiavoni G, Mattei F, Sestili P, et al. ICSBP is essential for the development of
mouse type I interferon-producing cells and for the generation and activation of
CD8alpha(+) dendritic cells. J Exp Med. 2002;196:1415-1425.
110. Aliberti J, Schulz O, Pennington DJ, et al. Essential role for ICSBP in the in vivo
development of murine CD8alpha + dendritic cells. Blood. 2003;101:305-310.
111. Tsujimura H, Tamura T, Ozato K. Cutting edge: IFN consensus sequence
binding protein/IFN regulatory factor 8 drives the development of type I IFN-
producing plasmacytoid dendritic cells. J Immunol. 2003;170:1131-1135.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
42
112. Izaguirre A, Barnes BJ, Amrute S, et al. Comparative analysis of IRF and IFN-
alpha expression in human plasmacytoid and monocyte-derived dendritic cells.
J Leukoc Biol. 2003;74:1125-1138.
113. Kerkmann M, Rothenfusser S, Hornung V, et al. Activation with CpG-A and
CpG-B oligonucleotides reveals two distinct regulatory pathways of type I IFN
synthesis in human plasmacytoid dendritic cells. J Immunol. 2003;170:4465-
4474.
114. Coccia EM, Severa M, Giacomini E, et al. Viral infection and Toll-like receptor
agonists induce a differential expression of type I and lambda interferons in
human plasmacytoid and monocyte-derived dendritic cells. Eur J Immunol.
2004;34:796-805.
115. Honda K, Yanai H, Negishi H, et al. IRF-7 is the master regulator of type-I
interferon-dependent immune responses. Nature. 2005;434:772-777.
116. Osawa Y, Iho S, Takauji R, et al. Collaborative action of NF-kappaB and p38
MAPK is involved in CpG DNA-induced IFN-alpha and chemokine production in
human plasmacytoid dendritic cells. J Immunol. 2006;177:4841-4852.
117. Schotte R, Nagasawa M, Weijer K, Spits H, Blom B. The ETS transcription
factor Spi-B is required for human plasmacytoid dendritic cell development. J
Exp Med. 2004;200:1503-1509.
118. Kawakami Y, Inagaki N, Salek-Ardakani S, et al. Regulation of dendritic cell
maturation and function by Bruton's tyrosine kinase via IL-10 and Stat3. Proc
Natl Acad Sci U S A. 2006;103:153-158.
119. Sochorova K, Horvath R, Rozkova D, et al. Impaired Toll-like receptor 8-
mediated IL-6 and TNF-alpha production in antigen-presenting cells from
patients with X-linked agammaglobulinemia. Blood. 2007;109:2553-2556.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
43
120. Jyonouchi H, Geng L, Toruner GA, Vinekar K, Feng D, Fitzgerald-Bocarsly P.
Monozygous twins with a microdeletion syndrome involving BTK, DDP1, and
two other genes; evidence of intact dendritic cell development and TLR
responses. Eur J Pediatr. 2007.
121. Avril T, Attrill H, Zhang J, Raper A, Crocker PR. Negative regulation of
leucocyte functions by CD33-related siglecs. Biochem Soc Trans.
2006;34:1024-1027.
122. Sjolin H, Robbins SH, Bessou G, et al. DAP12 signaling regulates plasmacytoid
dendritic cell homeostasis and down-modulates their function during viral
infection. J Immunol. 2006;177:2908-2916.
123. Takaki R, Watson SR, Lanier LL. DAP12: an adapter protein with dual
functionality. Immunol Rev. 2006;214:118-129.
124. Janssens S, Beyaert R. Functional diversity and regulation of different
interleukin-1 receptor-associated kinase (IRAK) family members. Mol Cell.
2003;11:293-302.
125. Pozzobon M, Marafioti T, Hansmann ML, Natkunam Y, Mason DY. Intracellular
signalling molecules as immunohistochemical markers of normal and neoplastic
human leucocytes in routine biopsy samples. Br J Haematol. 2004;124:519-
533.
126. Chu CL, Lowell CA. The Lyn tyrosine kinase differentially regulates dendritic cell
generation and maturation. J Immunol. 2005;175:2880-2889.
127. Cao W, Rosen DB, Ito T, et al. Plasmacytoid dendritic cell-specific receptor
ILT7-Fc epsilonRI gamma inhibits Toll-like receptor-induced interferon
production. J Exp Med. 2006;203:1399-1405.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
44
128. Zhang XM, Walsh B, Mitchell CA, Rowe T. TBC domain family, member 15 is a
novel mammalian Rab GTPase-activating protein with substrate preference for
Rab7. Biochem Biophys Res Commun. 2005;335:154-161.
129. Koumanov F, Holman GD. Thrifty Tbc1d1 and Tbc1d4 proteins link signalling
and membrane trafficking pathways. Biochem J. 2007;403:e9-11.
130. Matsumoto Y, Imai Y, Lu Yoshida N, et al. Upregulation of the transcript level of
GTPase activating protein KIAA0603 in T cells from patients with atopic
dermatitis. FEBS Lett. 2004;572:135-140.
131. Ito T, Amakawa R, Fukuhara S. Roles of toll-like receptors in natural interferon-
producing cells as sensors in immune surveillance. Hum Immunol.
2002;63:1120-1125.
132. Kadowaki N, Liu YJ. Natural type I interferon-producing cells as a link between
innate and adaptive immunity. Hum Immunol. 2002;63:1126-1132.
133. Uematsu S, Akira S. Toll-like receptors and Type I interferons. J Biol Chem.
2007;282:15319-15323.
134. Wang JP, Asher DR, Chan M, Kurt-Jones EA, Finberg RW. Cutting Edge:
Antibody-mediated TLR7-dependent recognition of viral RNA. J Immunol.
2007;178:3363-3367.
For personal use only. by guest on September 19, 2011. bloodjournal.hematologylibrary.orgFrom
