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Blood First Edition Paper, prepublished online June 3, 2011; DOI 10.1182/blood-2010-11-319699
BAD-LAMP is a novel biomarker of non-activated human
plasmacytoid dendritic cells
Axel Defays¶ 1,2,3, Alexandre David¶ 1,2,3, Aude de Gassart1,2,3,
Francesca De Angelis Rigotti 1,2,3, Till Wenger1,2,3,
Voahirana Camossetto1,2,3, Pierre Brousset4, Tony Petrella5, Marc Dalod1,2,3,
Evelina Gatti1,2,3,* and Philippe Pierre1,2,3,*
1
Centre d’Immunologie de Marseille-Luminy, Université de la Méditerranée, Case 906, 13288
Marseille cedex 9, France
2
INSERM, U631, 13288 Marseille, France
3
CNRS, UMR6102, 13288 Marseille, France
4
INSERM, U563, CPTP, 31024 Toulouse, France
5
Centre de Pathologie, 21000, Dijon, France
¶ both first authors contributed equally to this work
* both last authors contributed equally to this work
[email protected] or [email protected],
telephone: + 33 4 91 26 94 79, telefax: + 33 4 91 26 97 30
1
Copyright © 2011 American Society of Hematology
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Abstract
The brain and DC associated LAMP-like molecule (BAD-LAMP/c20orf103/UNC-46) is
a newly identified member of the family of lysosome-associated membrane proteins
(LAMPs). BAD-LAMP expression in mouse is confined to neurons. We demonstrate
here that in humans, BAD-LAMP can specifically be found in the type I interferonproducing plasmacytoid DCs. Human BAD-LAMP is localized in the ERGIC of freshly
isolated CD123+ pDCs and is rapidly lost upon activation by unmethylated cytosinephosphate-guanine (CpG) oligonucleotides. The restricted pattern of BAD-LAMP
expression allows for the rapid identification of normal and leukemic human pDCs in
tissues and blood.
Keywords: pDC, ERGIC,TLR9, UNC-47, c20orf103, CpG, UNC93B1
Running title: BAD-LAMP is a novel marker of Human pDCs
2
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Introduction
Plasmacytoid dendritic cells (pDCs) represent a rare but important cell type in the
hematopoietic system1,2,3. pDCs have been shown to be the principal cell type
producing type-I interferon (IFN) in response to viruses or during autoimmune
diseases3,4. In addition, pDCs can function as APCs during immune responses and
can promote antigen specific self-tolerance5,6. In humans, pDCs differ from
conventional dendritic cells (CD11c+ BDCA1+ myeloid DCs) as they uniquely
express Toll-like receptors (TLR) 7 and 97,8, which enable them to sense efficiently
endocytically-captured nucleic acids (e.g. CpG oligonucleotides)4,9,10,11.
Upon CpG ligation to TLR9, pDCs secrete high amounts of type I IFN and/or can
differentiate to acquire the ability to stimulate naïve T cells and to modulate the
immune response12,13. During differentiation, pDCs acquire antigen presentation
capacity, up-regulate MHC molecules, as well as a broad range of co-stimulatory
molecules14. Concurrently they also lose their type-I IFN production potential and
down-modulate innate immunity receptors, such as TLR9, ILT7 or BDCA-23,10. pDC
activation/differentiation
compartments,
induces
including
the
reorganization
endosomes.
Hence
the
of
different
expression
of
intracellular
molecules
participating to these changes could be specifically regulated upon pDCs
activation/differentiation.
Such regulation can be observed for TLR7 and TLR9, which reside mostly in the
endoplasmic reticulum (ER) of resting pDCs and, upon microbial activation, travel to
the endosomes to get proteolytically activated11. Several chaperone proteins are
involved in controlling TLR egress from the ER15,16. Among these, UNC93B1, a multi-
3
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transmembrane ER protein, specifically interacts with the transmembrane domains of
TLR3, TLR7 and TLR9 and controls their delivery to the endosomes upon
activation17,18. Mouse Unc93B1 mutant (3d) cannot signal via their intracellular
TLRs19 and in human patients Unc93B deficiency has been linked to the etiology of
herpes simplex virus-1 encephalitis20.
Human pDCs are generally identified with markers such as BDCA-4 (Neuropillin-1),
BDCA-2 (C-type lectin CLEC4C) and the IL-3 receptor α chain (CD123)21. However
these molecules are expressed by other hematopoietic cell types and are influenced
by the immunological context. BDCA-4 is up-regulated on activated myeloid DCs22
and CD123 is also expressed by basophils. Thus, the characterization of new
markers for human pDCs is important to improve their detection23. Exemplifying this
situation, a rare cutaneous tumor, termed blastic plasmacytoid dendritic cell
neoplasm (BPDCN), has been proposed to originate from pDCs, due to the
expression of molecular markers such as CD4, CD56, CD123, TCL1 and
CD2AP23,24,25,26,27. However, difficulties in diagnosis can arise, since these markers
are not unique to pDCs and sometimes aberrantly expressed by other cell types
present in tumors. There is therefore a strong need for additional and robust markers
of human pDC detectable in routine biopsies of neoplastic samples.
Via an in silico search for molecules involved in the organization of the endocytic
pathway, we identified a new member of the LAMP protein family: brain and DC
associated LAMP-like molecule (BAD-LAMP, c20orf103; UNC-46)28. BAD-LAMP is a
transmembrane glycosylated protein, which shares sequence and structural
homology with the canonical LAMP1 and LAMP2 molecules (CD107)29,30. BAD-LAMP
4
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harbors an endosomal addressing signal within is cytoplasmic tail, and contains
several conserved cysteine residues, which allow for the formation of particular
structural loops known as “LAMP folds”. Mouse BAD-LAMP was shown to
accumulate in a novel endocytic compartment in specific subtypes of cortical
projection neurons28. At functional level, mutations in UNC-46, the Caenorhabditis
elegans ortholog of BAD-LAMP cause, in the nematode, defects in most
neurotransmitter GABA-mediated behaviors. On this basis UNC-46/BAD-LAMP has
been proposed to act as a sorting chaperone addressing the membrane-associated
GABA transporter (UNC-47) to synaptic vesicles31.
Although human BAD-LAMP, like its murine homologue28, is principally expressed in
the brain, we show here that among blood cells, it is also specifically found in primary
CD123+ pDCs and BPDCN. BAD-LAMP mRNA and protein levels are downregulated upon CpG DNA stimulation of freshly isolated primary BDCA-4+ human
blood pDCs. In these cells, BAD-LAMP is mostly localized in the Endoplasmic
Reticulum-Golgi Intermediate Compartment (ERGIC) and like TLR9 its pattern of Nglycosylation remains endoglycosidase H-sensitive. Interestingly in HeLa cells,
ectopically expressed BAD-LAMP and UNC93B1 mutually influence their intracellular
localization and efficiently co-localize to a specific subset of late endosomes. Thus
BAD-LAMP represents a novel marker of human primary and transformed pDCs.
5
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Material and Methods
Bioinformatics and gene arrays.
Cell purification and RNA preparation as well as Gene array and meta-analysis were
performed as described previously32.
Molecular biology
Northern blot was done with FirstChoice Northern Blot Human Blot I (Ambion) using a
probe corresponding to exons 4, 5 and 6 of BAD-LAMP (clone IMAGE 6044324). The
cDNAs coding for BAD-LAMP were obtained from the IMAGE consortium. BADLAMP mutants and tagged forms were done as described previously28. The human
brain, spleen and skin total RNA extracts were obtained from Zyagen. The human
UNC93B1-His cDNA construct was a kind gift from Dr J-L Casanova (Rockfeller
University, NY, USA). The pUNO-TLR9-HA vector was obtained from Invivogen.
Antibodies and immunocytochemistry.
Monoclonal antibody 34.2 against BAD-LAMP was raised in rat against the peptide
“KMTANQVQIPRDRSQYKHM” corresponding to BAD-LAMP cytoplasmic tail. For
FACS analysis, 34.2 mAb was directly labeled with fluorochrome Cy5 using the Cy5
Ab Labelling kit from GE Healthcare. Anti-CD123 (AC145) and anti-BDCA-4
(AD517F6) antibodies were obtained from Miltenyi Biotec, anti-FLAG (M2) antibody
was from Sigma, anti-transferrin receptor was from Dr I. Mellman (New Haven, USA).
Rabbit anti-HA tag (9110), mAb anti-LAMP1 (H4A3), anti-KDEL (10C3) and anti-PDI
(RL90) were from AbCam, anti-His from Thermo Pierce, anti-CD63 (H5C6) and antiGM130 (35) from BD-Transduction, rabbit anti-HLA-ABC was from Dr J. Neefjes,
6
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(NKI, Amsterdam, NL) and anti-HLA-DR (XD5) from Dr J. Thibodeau (University of
Montreal, CA). All secondary antibodies were from Molecular Probes (USA), except
Cy3-5
secondary
antibodies,
which
were
from
Jackson
Immunoresearch.
Immunofluorescence and confocal microscopy was performed with a Zeiss LSM 510
as described previously33. Briefly for IF staining, pDCs and MoDCs were incubated
on 1% Alcyan blue-coated glass slides for 15 min and subsequently fixed in 3% PFA
for 15 min. ICC and IF staining were done in PBS, 10mM glycine, 5% FCS, 0.05 %
saponin. Human lymph node and tonsil sections were kindly provided by Dr Norbert
Vey,
Institut
Paoli
Calmettes,
Marseille.
Tissue
microarray
(TMA)
and
immuhistochemical analysis was performed as described previously34. Spleen cells
from humanized γc/RAG -/- mice were kindly provided by Dr. Sophie Ugolini (CIML,
Marseille). Image quantification and analysis was performed using the IMAGEJ
software (NIH, USA) and the JACoP pluggin28.
Cell purification and culture
Human PBMCs were isolated from whole blood by density gradient using FicollPaque PLUS (GE Healthcare). BDCA-4+ cells were magnetically sorted by positive
selection using MicroBeads kit and AutoMACS cell separator (Miltenyi Biotec). Sorted
cells were >95% pDCs based on BDCA-2 staining. pDCs were cultured at 0.5 to
1.106 cells/mL in RPMI-1640 containing 10% FCS and complemented with IL-3 at 10
ng/mL (unless stated otherwise). pDCs were stimulated with ODN 2216 (A-type),
ODN 2006 (B-type) or ODN M362 (C-type) at a concentration of 2.5 µM. CD14+ cells
were magnetically sorted by positive selection using MicroBeads kit and AutoMACS
cell separator (Miltenyi Biotec). Sorted monocytes were cultured at 2.106 cells/mL in
RPMI
1640
supplemented
with
10%
7
FCS,
nonessential
amino
acids,
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penicillin/streptomycin at 100 ng/ml and complemented with GM-CSF and IL-4 for 6
days for differentiation in MoDCs. HeLa cells were grown in DMEM containing 10%
FCS supplemented with penicillin/streptomycin at 100 ng/mL.
Cell transfection
HeLa cells were seeded the day before transfection at a cell concentration of 2.105
cells/mL. Transfections were performed using Lipofectamine 2000 reagent. Cells
were harvested 24h after transfection for lysis. HeLa cells used for IF were seeded
on microscopy glass slides before transfection and fixed in 3% PFA for 15 min 24h
after transfection. MoDCs were transfected at 5 days of differentiation using in vitro
transcribed mRNA as described previously35.
RT-PCR and mRNA extraction
RNA extraction was performed with the RNeasy Mini kit (Qiagen) except for human
spleen FirstChoice total RNA (Ambion). RT-PCR was performed using Superscript II
enzyme (Invitrogen) for the reverse transcription and Taq polymerase (Invitrogen) for
the PCR amplification. PCR amplification was performed for 30 cycles unless stated
otherwise. Quantitative RT-PCR was done using SYBR Green PCR buffer (PE
Biosystems) as described previously35 and analysis of the results were obtained with
REST software36.
Immunoblots and immunoprecipitation
1% Triton X-100 cell extracts complemented with protease inhibitors cocktail (Roche)
and 5 mM MG132 (Sigma) were immunoblotted after separation by 12% SDS-PAGE.
Immunoprecipitation was performed with 5 µg/sample of 34.2 antibody and protein G-
8
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Agarose beads (Millipore). Endoglycosidase H (Calbiochem) treatment was
performed as described33.
Results
Human c20orf103/BAD-LAMP/UNC-46 mRNA is expressed in pDCs
Affymetrix Human Genome U133 Plus 2.0 arrays and Mouse Genome 430 2.0 were
used to generate gene expression profiles of human blood monocytes, neutrophils, B
cells, NK cells, CD4 or CD8 T cells and 18 mouse leukocyte profiles32. Our data were
complemented with public databases on human blood DC subsets (pDCs, BDCA-1
cDCs, BDCA-3 cDCs, and lin-CD16+HLA-DR+ cells). Comparing mouse and human
hematopoietic cell compendia, we identified BAD-LAMP/C20orf103 as a molecule
expressed specifically in human pDCs among other hematopoietic cells (Figure 1A).
At nucleotide level, the human BAD-LAMP sequence is homologous at 45% with
human LAMP 1 and LAMP 2, the firstly identified members of the LAMP family. BADLAMP mRNA codes for a protein of 280 aa, (PI 6.42 and MW 31.7 kDa) predicted to
contain a transmembrane domain (aa 236-256) and a 24 residues cytoplasmic tail
(Figure S1A). The cytoplasmic domain contains a YKHM sequence (aa 276)
corresponding to a classical YXXΦ internalization and endosomal targeting motif.
The luminal domain contains 4 highly conserved cysteine residues, separated by an
amino acid stretch of a length compatible with the formation of stable di-sulfide bonds
and the acquisition of a classical “LAMP fold”. Human BAD-LAMP is 85% identical at
amino acid level to its murine homologues and was also predicted to contain 3
characteristic N-glycosylation sites.
9
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A more global analysis of microarrays data sets available for different organs and cell
types32 confirmed that human BAD-LAMP is expressed at similar levels and above
the detection threshold only in different brain tissues and in pDCs (Figure S2). This
conclusion was suported by Northern blot analysis of different human tissues, during
which BAD-LAMP mRNA was exclusively detected in the adult brain (Figure 1B), as
a transcript of around 1.8 kb with no apparent alternative spliced forms. To
determine, if BAD-LAMP mRNA was truly expressed in pDCs, we first carried-out a
RT-PCR on total human spleen mRNA and failed to reveal the presence of any
specific BAD-LAMP transcript (Figure 1C). We could however amplify successfully
BAD-LAMP messenger from the same mRNA extract by nested PCR, a result never
observed when performed with mouse BAD-LAMP specific primers and mouse
spleen mRNAs (not shown). The low level of detected BAD-LAMP mRNAs in human
spleen was likely to reflect the rareness of pDCs in this organ, which are certainly in
insufficient numbers to reveal BAD-LAMP transcription by tissue Northern blot. These
results also supported our gene expression analysis, excluding BAD-LAMP
expression from mouse leukocytes and lymphoid organs (Figure S3). We next
attempted to confirm BAD-LAMP expression in different tissues and isolated cells
using semi quantitative nested RT-PCR. As anticipated from the microarray data, we
could amplify BAD-LAMP transcript from total human brain and spleen mRNAs, as
well as from material isolated from magnetically immuno-purified blood BDCA-4+
-
pDCs (Figure 1D). Control mRNAs isolated from BDCA4 leukocytes and HeLa cells
was found negative for BAD-LAMP expression. Surprizingly, we could amplify BADLAMP transcript from total skin mRNAs (Figure 1D), an organ in which BAD-LAMP
mRNA expression was considered below the significance threshold in the examined
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microarray data compendium (Figure S2).
Human BAD-LAMP is expressed in CD123/BDCA-2 pDCs.
In order to detect BAD-LAMP expression as a protein, a monoclonal antibody (mAb
34.2) was raised against the last 12 amino acids of BAD-LAMP cytoplasmic tail
(Figure S1A). This antibody recognized efficiently by immunofluorescence confocal
microscopy the eGFP-tagged version of BAD-LAMP ectopically expressed in HeLa
cells (Figure S1B). By immunohistochemistry (IHC) performed on human spleen and
tonsil sections (Figures 2A and S4A, S4B and S4C), BAD-LAMP was detected in a
rare cell type also positive for the two markers CD123 and CD4 and often found in
the vicinity of high endothelial venules, a characteristic localization for pDCs1,2,3.
Interestingly at high magnification, CD123 staining looked also intracellular, a
situation also partially observed in isolated pDCs (not shown), but considerably
enhanced by the epitope revealing treatment used to process the paraffin sections.
We next performed FACS on human peripheral blood monocytes (PBMC) using Cy5conjugated 34.2 mAb. A rare BDCA-4 and BDCA-2 positive population of blood cells
(0.34%), likely to represent circulating pDCs, was singled-out by 34.2 intracellular
staining (Figure 2B). BAD-LAMP expression in pDCs was further confirmed by the
+
detection of an homogenous positive labelling of blood BDCA-4 cells (>95% pDCs)
(Figure 2C). Moreover, confocal microscopy experiments, performed on the same
freshly isolated pDCs, indicated that BAD-LAMP accumulates mostly in intracellular
membrane compartments (Figure 2C).
BAD-LAMP mRNA being undetectable in mouse leukocytes both by gene arrays and
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RT-PCR, we attempted to visualize BAD-LAMP expression in human pDCs isolated
from the spleen of γc/RAG -/- mouse reconstituted with human CD34+ hematopoietic
stem cell37. Confocal microscopy performed in parallel with anti-BDCA-4 and 34.2
revealed the presence of rare double positive human splenocytes in “humanized”
mouse spleen (Figure S5). This result confirmed that human pDCs differentiation is
supported efficiently in CD34+ reconstituted mice and that BAD-LAMP can be used
as a marker to track this rare cell type in different human biological samples.
IL-3- and CpG-induced maturation decrease BAD-LAMP levels.
We studied BAD-LAMP protein expression in cell extracts obtained from human
pDCs, PBMCs, monocyte-derived DCs (MoDC) and HeLa cells transfected with BADLAMP cDNA (Figure 3A). By immunoblot, pDCs were the only hematopoietic cell type
found to express naturally a 35 kDa form of the molecule. In transfected HeLa cells,
used here as control, we detected several additional glycosylated forms absent from
the pDC extract28. We then asked if pDC activation could influence BAD-LAMP
expression. Purified BDCA4+ cells were cultivated with IL-3 in presence of different
types of CpG oligonucleotides, known to promote IFN-type I secretion. Upon
exposure to CpG, a strong diminution in BAD-LAMP mRNA levels was observed
(Figure 3B). This decrease was progressive over 24h and independent from the type
of CpG used in the experiment. Interestingly BAD-LAMP levels were affected by IL-3
treatment alone (Figure 3B), confirming that IL-3 is able to induce pDC activation
independently of TLR signaling3. Both by intracellular FACS and immunoblot
quantification, BAD-LAMP protein levels were found to be steadily reduced during
CpG-mediated activation, indicating that BAD-LAMP is mostly expressed in nonactivated human pDCs and lost upon nucleic acid detection (Figures 3C and 3D).
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BAD-LAMP is expressed in blastic plasmacytoid dendritic cell neoplasm
pDC neoplastic transformation gives rise to the recently described BPDCN
pathology27. At the morphological level, skin biopsies show a monomorphous cell
proliferation simulating a pleomorphic T cell cutaneous lymphoma. The diagnosis of
this neoplasm is mostly based on phenotypic criteria, namely histological analysis of
tissue sections. Currently, the characterizing features of BPDCN are the expression
of CD4, CD56 and CD123 antigens, and the absence of lineage specific markers for
B-cell, T-cell, NK-cell and myeloid-cell lineages. Our characterization of BAD-LAMP
as a non-activated human pDC-specific marker led us to explore whether the
detection of this molecule could facilitate the diagnostic of this rare tumor. We
examined multiple paraffin sections from CD4+/CD56+/CD123+ tumors and could
show that almost all were strongly stained by the 34.2 monoclonal antibody (Figure
4A). Analysis by tumor protein arrays (TPA) of 33 different tumors, classified as
BPDCN, revealed that 78% stained positively for BAD-LAMP (Figure 4B).
Interestingly, BAD-LAMP was not expressed in any of the other hemato-malignancies
tested, including B and T lymphomas (supplementary table 1). In different histological
analysis, we could also observe a light BAD-LAMP staining in supra-basal skin
keratinocytes, which was considerably increased in squamous cells (Figure S6A and
S6C). BAD-LAMP staining clearly intensifies in the vicinity of the stratum
corneum, and since this area is often associated with non-specific staining,
BAD-LAMP heterogenous reactivity in keratinocytes could reflect more their
differentiation state, than a true expression of the molecule. Consequently, the
PCR amplification of BAD-LAMP transcripts from total human skin mRNAs could
either indicate the presence of rare resident pDCs in the skin samples used to
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prepare the RNA or reflect the contribution of a weak BAD-LAMP expression in some
keratinocytes. Importantly, further histological analysis indicated the BAD-LAMP
expression was not influenced by the state of skin inflammation as judged from the
observation of non-specific dermo-epidermitis biopsies (Figure S6B). Importantly, this
light epithelium staining was easily distinguishable morphologically from the BADLAMP positive detection obtained in BPDCN. BAD-LAMP represents therefore a
novel and relevant marker for blastic plasmacytoid dendritic cell neoplasm, improving
significantly the histological characterization of these tumors by a single round of
staining.
BAD-LAMP is addressed in the ERGIC of pDCs and ER of transfected MoDCs
In a previous report28, the study of BAD-LAMP intracellular localization in mouse
neurons has allowed us to define a non-conventional early endosomal compartment.
We tried to establish if in primary pDCs, its sub-cellular distribution would coincide
with the neuronal one (Figure 5A and S7). Confocal immunofluorescence microscopy
revealed that BAD-LAMP accumulates in a vesicular pattern distinct from the staining
obtained with HLA-DR, HLA-A and GM130 (Golgi) (Figure S7). Staining performed
with early (transferrin receptor) and late endocytic markers (CD63 and LAMP1) also
failed to show any obvious co-localization with BAD-LAMP (Figure 5A and S7),
confirming that the molecule does not accumulate in late endosomes, nor at the
plasma membrane.
BAD-LAMP however, displayed a significant degree of co-
localization with the Endoplasmic Reticulum-Golgi Intermediate Compartment
(ERGIC) ERGIC-53 (Figure 5A), suggesting that BAD-LAMP resides mostly in the
ERGIC of pDCs, and surprisingly not in the ER of these cells as judged by its existing
but weak co-localization with KDEL-bearing molecules (Figure S7B). We further
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evaluated biochemically BAD-LAMP distribution by establishing the type of Nglycosylation acquired by the molecule during its intracellular transport. After
immunoprecipitation, exposure to glycanases and revelation by immunoblot, we
found that the bulk of BAD-LAMP molecules remained endoglycosidase H-sensitive
(Figure 5B), confirming that it mostly resides in a pre-Golgi compartment (such as
ERGIC), and does not use the classical secretory pathway for further potential
transport, as previously observed for TLR receptors or UNC93B1 transport18. The
loss of BAD-LAMP triggered by CpG stimulation was very rapid, and its intracellular
distribution upon pDC activation, even at early stages (1 and 6h), could not be
efficiently established (Figure 5C). This situation is extremely different from neurons
or transfected HeLa cells in which BAD-LAMP accumulates respectively in a subset
of endosomes or at the plasma membrane28. This complex and variable distribution
among cell types suggests a possible interaction of BAD-LAMP with other molecules
expressed specifically in distinct cell subsets and capable of interfering with its
intracellular transport. This hypothesis was further strengthened, by the extended colocalization of the ER-resident proteins disulfide isomerase (PDI) and BAD-LAMP
observed upon the transfection of BAD-LAMP mRNA in human monocyte-derived
DCs (Figure 5D), indicative of an ER accumulation previously not observed for this
molecule.
BAD-LAMP and UNC93B1 are co-localized upon transfection in HeLa cells.
In Caenorhabditis elegans, UNC-46 has been shown to interact with vesicular GABA
transporter (UNC-47) and promote its co-targeting to synaptic vesicles, supporting a
potential chaperone role for BAD-LAMP/UNC-46 through specific interactions with
other transmembrane proteins. Interestingly, a yeast two-hybrid screen performed
with C. elegans proteins has revealed a direct interaction between UNC-46 and an
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UNC-93 related protein, F31D5.238. Since, UNC93B1 is highly expressed in human
pDCs2 compared to other leukocytes (Figure S8A), we decided to investigate
whether BAD-LAMP could interact with the endosomal TLR-chaperone.
We chose HeLa cells as an experimental system, since they do not express
UNC93B1, TLR9, nor BAD-LAMP, so that we could follow the intracellular transport
of different tagged-versions of these molecules expressed individually or in
combination (Figure S8B). The distribution of the different molecules was examined
by confocal microscopy (Figures 6 and S9). As previously shown28, untagged BADLAMP and N-terminally FLAG-tagged BAD-LAMP (flagBAD) were mostly found
accumulating at the plasma membrane and more rarely in transferrin-positive
endosomes (Figure S9), The mutation of the cytoplasmic tail tyrosine residue 276 to
alanine restricted the flagBADY276A protein to a complete cell surface distribution, due
to a defect in its internalization and recycling28 (Figures S9, 6A and C). Interestingly,
a flagBAD mutant completely deleted of its cytosolic tail (flagBAD-ΔCt), was found
almost uniquely in the ER of transfected cells co-localizing with PDI (Figures S9 and
6C), suggesting that BAD-LAMP cytoplasmic domain is also important for its ER
export. Conversely, when an eGFP moiety was fused C-terminally to BAD-LAMP
(gpfBAD), the resulting chimera was mostly addressed to LAMP1+ late endosomes
and lysosomes (Figure 7). This abnormal sorting of gfpBAD indicates, that a profound
structural modification of BAD-LAMP cytoplasmic tail or its potential dimerization
induced by eGFP can enhance the capacity of BAD-LAMP to reach and to remain
associated with late endosomal compartments under specific circumstances.
UNC93B1 was expressed as a 6xHIS-tagged form (hisUNC). Accordingly to previous
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work, hisUNC expressed alone accumulated in the ER of transfected HeLa cells17
(Figure 6A). Co-expression of flagBAD and hisUNC provoked a redistribution of the
two molecules and their co-localization in bright punctuate intracellular structures,
likely to be endosomes (Figure 6B). The same phenotype was obtained for gfpBAD
and hisUNC co-expression, confirming the nature of the targeted compartment as
LAMP1+ endosomes (Figure 7). This effect was BAD-LAMP-specific since coexpression of the related lysosome-associated protein, DC-LAMP, with UNC93B1 did
not have any effect on the sub-cellular distribution of the TLR chaperone, which
mostly remained in the ER (Figure 6B). BAD-LAMP cytoplasmic tail, but not its YXXΦ
motif, seemed important for efficient co-chaperoning, since, when co-expressed with
hisUNC, flag-BADY276A no longer distributed entirely to the plasma membrane and
was able to support UNC93B1 endosomal targeting (Figure 6C). On the contrary,
flagBAD-∆Ct co-expression with hisUNC had a modest impact and only a small
portion of the two molecules could be found in endosomal compartments, while the
bulk remained in the ER (Figure 6C). This active and efficient intracellular relocalization upon co-expression of the two molecules indicates that UNC93B1 and
BAD-LAMP could function as co-chaperones and have a reciprocal influence on their
intracellular addressing.
Discussion
Human BAD-LAMP represents a new member of the LAMP family, based on
sequence analysis. However its tissue expression pattern and intracellular
distribution are quite unusual compared to other classical LAMP family members,
which have a widespread expression and specifically accumulate late endosomes
and in lysosomes. In mouse, BAD-LAMP is mostly expressed in brain while in human
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it is also found in CD123+/BDCA2+/BDCA4+ plasmacytoid dendritic cells circulating in
the blood or localized in secondary lymphoid organs. This situation is reminiscent of
the tissue distribution of another non-conventional LAMP family member, DCLAMP/LAMP3, which is expressed both in activated human conventional DCs and in
human type II pneumocytes39, while its expression remains restricted to type II
pneumocytes in mouse40.
Blastic plasmacytoid dendritic cell neoplasm, which was previously called
CD4+/CD56+ hematodermic neoplasm and blastic NK-cell lymphoma, is a
hematopoietic malignancy of pDC origin. The recent discovery of CD123 and BDCA2 expression in BPDCN has been determinant to point towards its pDC origin23,25,26.
Clinically, most cases of CD4+/CD56+ leukemia show initial cutaneous involvement,
although pDCs are generally absent from normal skin. Our discovery of BAD-LAMP
expression in these tumors definitely confirms their plasmacytoid origin and suggests
that these neoplastic cells are in a resting state. Indeed, BAD-LAMP is only
expressed abundantly in freshly isolated pDCs and its expression is lost upon
activation by TLR ligands. A majority of leukemic pDCs are therefore phenotypically
similar to their normal resting counterparts and BAD-LAMP detection offers an novel
and alternative mean of identifying these aggressive tumors. 20% of the tested
BPDCN being negative for BAD-LAMP, it will be of interest to determine if this lack of
expression pinpoints a specific category of neoplasms, which are characterized by a
different activation state or fall in a different clinical cohort.
In a previous report28, we showed that in neurons, BAD-LAMP was mostly addressed
in a subset of endosomal structures accumulating in the growth cone. We show here
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that in pDCs, BAD-LAMP accumulates in the ERGIC, prior to its disappearance upon
activation by CpG nucleotides sensing. Interestingly UNC-46, the C. elegans ortholog
of BAD-LAMP, has been shown to serve as a chaperone for the GABA transporter
(UNC-47) molecule and to be required to sort properly the transporter in synaptic
vesicles31. UNC-47 has also been shown to influence reciprocally the traffic of UNC46, suggesting the existence of a co-chaperoning mechanism allowing the two
molecules to exit together from the ER and reach synaptic vesicles. Interestingly,
although many neuronal molecules are found in pDCs (eg. BDCA-4/Neuropilin-1 or
Pacsin 1/syndapin)32, no significant expression of the GABA transporter could be
detected in these cells, further suggesting that BAD-LAMP could serve as a cochaperone for other transmembrane molecules expressed in human pDCs, and
potentially not in neurons.
The discovery of an interaction between UNC-46 and the UNC-93-related protein
F31D5.2, led us to investigate if the UNC-93 ortholog, UNC93B1, which is expressed
in high amount in human pDCs compared to other cells (Figure S8A and
http://biogps.gnf.org), could be one the molecule exerting a co-chaperoning activity
with BAD-LAMP. In HeLa cells, which are not expressing UNC-93B1, nor the GABA
transporter, BAD-LAMP is misrouted to the cell surface, but can reach late
endosomes, upon co-expression with UNC-93B1. Alternatively in MoDCs, which
naturally express UNC-93B1 (Figure S8A), BAD-LAMP is retained in the ER and
does not display any obvious endosomal localization, even when DCs are stimulated
though TLR3, which also interacts with UNC93B1 (not shown). Thus although BADLAMP can be endocytosed and recycled through a tyrosine-based addressing signal
within its cytoplasmic tail28, its intracellular transport seems to depends on factor
19
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expressed specifically in particular cell types and potentially at different sub-cellular
location, such as UNC-47 in neurons. The co-expression of BAD-LAMP and UNC93B1 in HeLa cells is artificial and several additional molecules interacting with UNC93B1 or BAD-LAMP are likely to be present in a physiological situation. UNC-93B1
distribution and the availability of other factors in BAD-LAMP expressing cells (e.g.
UNC-47 or TLR9) could influence BAD-LAMP transport or reciprocally be influenced
by BAD-LAMP. Therefore independently of considerations about the regulation of
UNC93B1 and endocytic TLRs trafficking, these data primarily suggest that BADLAMP has the ability to interact with multispan membrane proteins and is likely to
function as a co-chaperone in non-activated human pDCs and neurons.
Acknowledgements
We thank for expert technical assistance the PICsL imaging core facility and Michel
Pierres at the CIML monoclonal antibody facility. J-L Casanova for the kind gift of
reagents. This work is supported by grants to PP from Agence Nationale de la
Recherche (BAD-LAMP layers), La Ligue Nationale Contre le Cancer and l’ANRS.
ADe and ADa are supported by fellowships from MENRT, PACA and LNCC. PP is
part of the Sybaris FP7project. A. Defays, A. David, A. de Gassart, F. De Angelis
Rigotti, T. Wenger and V. Camossetto all performed research and analyzed data.
Pierre Brousset and T. Petrella performed research and analyzed TMA. M. Dalod
performed research and analyzed microarrays. E. Gatti and P. Pierre designed the
research, analyzed data and wrote the paper.
20
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Conflict of interest disclosure:
The authors declare to have no relevant financial conflict of interest.
21
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Figure legends
Figure 1: BAD-LAMP mRNA expression profile in Human
A. Gene microarray quantitation of BAD-LAMP mRNA expression in human
leukocytes. Results are shown as fluorescent signal intensity for Affymetrix Human
Genome U133 PLUS 2.0 ProbeSet 219463_at (expressed in arbitrary units in log
scale). Quality controls, data sources and data normalization are described in
Robbins SH et al., Genome Biology. 2008. Neu: neutrophils; pMφ: peripheral blood
mononuclear cell-derived macrophages; Mo-Mφ: monocyte-derived macrophages;
Mo-DC: monocyte-derived GM-CSF+IL-4 DC; CD16 DC: blood Lin-HLA-DR+CD16+
DC; BDCA1 DC: blood BDCA-1+ DC; BDCA3 DC: blood BDCA-3+ DC; pDC: blood
plasmacytoid DC; BL: blood B lymphocytes; CD4 TL: blood CD4+ T lymphocytes;
CD8 TL: blood CD8+ T lymphocytes; NK cells: blood natural killer cells. B. Tissue
expression of BAD-LAMP assessed by Northern Blot. A signal is detected only in
adult human brain. Actin mRNA levels are shown as control. C. Detection of BADLAMP transcript in human spleen RNA total extracts by nested RT-PCR. D. Detection
of BAD-LAMP transcript in total RNA extracts from human samples. Actin levels after
the first PCR round are shown as control. A plasmid containing BAD-LAMP cDNA
was used as a positive control.
Figure 2: BAD-LAMP is detected specifically in pDCs
A. Detection of BAD-LAMP in human lymphoid tissue. Paraffin human spleen
sections were stained with monoclonal antibodies against CD123 (red) and BADLAMP (green). Overlay show that BAD-LAMP+ cells are also CD123+ (merge, yellow).
25
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Bar 20µm. Paraffin-fixed human tonsil sections were stained in IHC (lower right).
BAD-LAMP+ cells display a pDCs morphology (arrows, x800) next to HEV. B.
Intracellular FACS staining on human PBMCs. A rare cell population can be isolated
based on BAD-LAMP expression (left). BAD-LAMP+ cells were identified as pDCs
based on BDCA-4 expression (right). C. BAD-LAMP staining on purified pDCs. Cells
stained in intracellular FACS (left) are homogenously BAD-LAMP+ (solid line) as
compared to isotype control background (filled graph). BAD-LAMP is localized in
intracellular membrane compartments of BDCA-4+ pDCs as shown by confocal
microscopy (green, right). Nucleus (Nu) staining is shown in blue. Bar: 20µm.
Figure 3: Regulation of BAD-LAMP during pDCs activation
A. BAD-LAMP detection by immunoblot. Cell lysates from different cell types were
separated by SDS-PAGE and revealed using the 34.2 mAb against BAD-LAMP. A
single specific band is detected in pDC extracts around 35 kDa and not in immature
monocyte-derived dendritic cells (MoDCs i), LPS-activated MoDCs (MoDCs m) nor in
total PBMCs. HeLa cells transfected with BAD-LAMP cDNA (HeLa BAD) and control
(HeLa nt) were used as a positive control both for specificity and as a reference for
the glycosylation pattern. Asterisk (*) marked lanes were loaded with a lower amount
of total proteins to compensate for the high BAD-LAMP expression levels in
transfected cells. Actin levels are shown as loading controls. B. BAD-LAMP mRNA
levels are down-regulated upon IL-3 treatment and CpG activation. Purified pDCs
were cultivated in presence of IL-3 for 6h or 24h and stimulated or not with A-, B- or
C-type CpG ODNs. BAD-LAMP mRNA levels were determined using quantitative RTPCR. Levels for CpG-treated cells were normalized relative to the IL-3 only condition.
Results are from one representative experiment (n=3). C. BAD-LAMP is down-
26
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regulated at the protein level upon CpG activation. After 24h of culturing freshly
isolated pDCs (filled graph) with IL-3 (solid black line) and A-type CpG ODN (dashed
gray line), BAD-LAMP expression monitored by intracellular FACS staining is downregulated pDCs. IL-3 treatment is sufficient to decrease BAD-LAMP levels. D. BADLAMP is no longer detectable by immunoblot in pDCs after 24h of A-type CpG ODN
stimulation. Low amounts of HeLa BAD and HeLa nt (*) were used as specificity
control. Actin levels are shown as loading controls.
Figure 4: BAD-LAMP is a marker of blastic pDC neoplasms
A. IHC on paraffin sections of skin lesions from patients with BPDCN reveal a
massive infiltration of BAD-LAMP+ cells (arrows, x400), negative staining with mouse
IgG isotype control is shown on the right. B. A larger scale analysis by tissue arrays
revealed that >78% of biopsies were BAD-LAMP+ among 33 patients diagnosed with
a CD4+/CD56+ malignancy (left). An example of a BAD-LAMP+ biopsy from the tissue
array is shown (right).
Figure 5: BAD-LAMP is localized in the ERGIC
A. Immunofluorescence staining for BAD-LAMP in purified pDCs. BAD-LAMP (green,
upper panels) co-staining with early endosomal marker transferrin receptor (TfR, red)
and lysosomal marker LAMP1 (blue) show no overlap. BAD-LAMP (green, lower
panels) and ERGIC marker ERGIC53 (red) display a strong co-localization (arrow)
confirmed by Pearson coefficient calculation (0.6). Bar: 10µm. B. Analysis of BADLAMP glycosylation by enzymatic treatments. Immunoprecipitation from pDC lysate
and subsequent endoglycosidase H (EndoH) treatment reveals that BAD-LAMP
glycosylation remains endo H-sensitive. Total lysate and antibodies alone (Ab) are
27
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shown as controls. C. Immunofluorescence staining for BAD-LAMP in pDCs cultured
with IL-3 + A-type CpG ODN for 6h. BAD-LAMP (green) is lost while ERGIC53
distribution (blue) is not affected by activation. Bar: 10µm. D. Confocal microscopy of
BAD-LAMP heterologous expression in human monocyte-derived DCs. 6h after
transfection, BAD-LAMP (green) and endoplasmic reticulum resident PDI (red)
display extensive co-localization. Bar: 10µm. Pearson’s coefficient values are given
as R=0.xxx.
Figure 6: BAD-LAMP co-localizes with UNC93B1 in transfected HeLa cells
A.
BAD-LAMP and UNC93B1 have different cellular localization when over-
expressed together in HeLa cells. BAD-LAMP (green, top) is mainly targeted to the
plasma membrane with a small portion is found in endocytic compartments.
UNC93B1 (red, bottom) is localized in the ER. Bar: 20µm. B. When co-expressed,
BAD-LAMP (green) and UNC93B1 (red) co-localize in large endosomal intracellular
vesicles (upper panels, arrows). On the contrary, upon expression of the structurally
related endosomal resident DC-LAMP (green) intracellular trafficking UNC93B1 (red)
remains unchanged (lower panels). C. Flag-tagged BAD-LAMP mutants have
different sorting behaviors. Flag-BAD-LAMP (wt) is targeted to the cell surface and
partially to endosomes (green, left panels), while the Flag-BAD-LAMP Y276A mutant
is almost exclusively localized at the plasma membrane. Flag-BAD-LAMP ΔCt mutant
is retained in the endoplasmic reticulum. Upon co-transfection with His-UNC93B1
(red, right panels), all the different flag-tagged forms of BAD-LAMP (green, right
panels) are sorted together with His-UNC93B1 (red) in the same intracellular
endosomal compartments (arrows). Pearson’s coefficient values are given as
R=0.xxx.
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Figure 7: BAD-LAMP-dependent sorting of UNC93B1 to the endosomes.
Immunofluorescence confocal microscopy of HeLa cells transfected with an eGFPtagged BAD-LAMP fusion. BAD-LAMP-GFP (green) is sorted to intracellular
compartments that are mostly LAMP1+ (blue, arrow, (upper panels). In cells cotransfected with BAD-LAMP-GFP and UNC93B1 (red, lower panels), the two
molecules are sorted together in LAMP1+ intracellular compartments. Pearson’s
coefficient values are given as R=0.xxx.
29
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Prepublished online June 3, 2011;
doi:10.1182/blood-2010-11-319699
BAD-LAMP is a novel biomarker of non-activated human plasmacytoid
dendritic cells
Axel Defays, Alexandre David, Aude de Gassart, Francesca De Angelis Rigotti, Till Wenger, Voahirana
Camosseto, Pierre Brousset, Tony Petrella, Marc Dalod, Evelina Gatti and Philippe Pierre
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