|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2647-2657
Expression of pT mRNA in a Committed Dendritic Cell Precursor in
the Human Thymus
By
Pieter C.M. Res,
Franka Couwenberg,
Florry A. Vyth-Dreese, and
Hergen Spits
From the Division of Immunology, The Netherlands Cancer Institute,
Antoni van Leeuwenhoek Huis, Amsterdam, The Netherlands.
 |
ABSTRACT |
We have characterized dendritic cell precursors (pre-DC) in the
human thymus. These
CD1a CD3CD4+CD8
cells express high levels of interleukin-3R (IL-3R) on the membrane and are able to develop into mature DC upon culture with IL-3
and CD40 ligation. The DC precursors are predominantly located in
the thymic medulla. Interestingly, the pre-DC express pT mRNA, which
is also present in
CD1a+CD3CD4+ CD8
pre-T cells. Yet, the pre-DC lack expression of recombination activating gene-1 mRNA and fail to develop into T cells in
appropriate assays. The thymic pre-DC are very similar to the recently
characterized pre-DC found in the T cell areas of the tonsil, and it is
suggested that these pre-DC populations are of lymphoid origin.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
DENDRITIC CELLS (DC) ARE highly
specialized antigen-presenting cells present in peripheral lymphoid and
nonlymphoid organs. These cells play an essential role in the
initiation of immune responses.1,2 DC are also present in
the thymus, where they serve to eliminate potentially autoreactive
cells from the T-cell repertoire.3 It has become clear that
there are distinct subsets of DC in the mouse with different
ontogenetic origins. While many DC in the periphery are of myeloid
origin, a second subset, which includes thymic DC, appears to be
related to lymphocytes.4-6 The notion that thymic DC are
related to lymphocytes is based on studies of Ardavin et
al,4 which demonstrated that DC in the mouse thymus are
derived from an intrathymic precursor with the capacity to develop into
natural killer (NK) and T cells, but not into myeloid cells. Recently,
a more mature precursor population in the mouse thymus was shown
to have T and DC differentiation capacity.7 Others
and we have shown that the human thymus contains precursors able to
develop into T, NK, and DC,8,9 but not into
monocytes,10 suggesting that also in man thymic DC may be
of lymphoid origin. Therefore, analysis of thymic DC may provide insight into the characteristics of human DC.
So far our knowledge about mature thymic DC and their immediate
precursors in man is limited. Sotzic et al11 have described a CD4+ population in the human thymus, which is able to
differentiate into DC after overnight culture. These cells expressed
relatively high levels of major histocompatability complex (MHC) class
II antigens, but lacked CD1a, which is expressed on Langerhans cells in
the skin. It is unclear whether this population are committed DC
precursors, represent already matured DC, or is a mixture of mature DC
and their immediate precursors. Here we have analyzed the
CD1aCD3CD4+CD8
population in the human thymus in more detail with emphasis on features
that these cells might share with T-cell precursors. We found that
these cells mature to DC upon coculture with interleukin-3 (IL-3) and
CD40 ligation. The thymic DC precursors share characteristics with
pre-T cells, including expression of pre-T cell receptor alpha chain
(pT ) mRNA, strongly suggesting their relationship with T cells.
However, these DC precursors are committed to the DC lineage, as they
are unable to develop into T and NK cells in appropriate assays.
Interestingly, the phenotype and precursor activities of these cells
are very similar to that of a committed DC precursor in the tonsil,
which was previously referred to as plasmacytoid T cell.12
| |
MATERIALS AND METHODS |
Isolation of DC precursors from tonsil and thymus.
Thymocyte tissues were obtained from children ranging from 3 weeks to 3 years of age undergoing median sternotomy and corrective cardiovascular
surgery. Tonsils had been removed from children for therapeutic
purpose. Suspensions were made by mincing tissues and pressing them
through a stainless steel mesh. Large aggregates were removed and the
cells were washed once before separating subpopulations. Thymocytes
were kept at 4°C overnight followed by centrifugation over a ficoll
gradient. The ficoll interphase cells were collected and depleted for
CD8+ cells by staining with RPA-T8 (kindly
provided by G. Aversa, DNAX, Palo Alto, CA) and subsequent negative
depletion using Dynal magnetic beads (Dynal, Oslo, Norway). Remaining
thymocytes were stained with anti-CD3 fluorescein isothiocyanate
(FITC), anti-CD8 FITC (which recognizes a different epitope on CD8 than
RPA-T8 used for depletion), anti-CD1a phycoerythrin (PE)
and anti-CD4 Tricolor monoclonal antibodies (MoAbs) and the
CD1aCD3CD4+CD8
DC precursors and the
CD1a+CD3CD4+CD8
immature single positive (CD4 ISP) thymocytes were isolated by sorting
on a FACStar plus (Becton and Dickinson, San Jose, CA). In some
experiments CD1aCD4+CD3+
mature thymocytes were sorted after staining of total thymocytes with
CD3 FITC, CD1a PE and CD4 Tricolor.
Single cell suspensions of tonsillar cells were labeled with anti-CD3,
anti-CD8 (RPA-T8), anti-CD14, anti-CD19 (CLB, CD14 and CD19,
respectively, gifts from Dr R. van Lier, CLB, Amsterdam, The
Netherlands) and anti-CD56 (L185, from J.H. Philips, DNAX, Palo Alto,
CA) and labeled cells were subsequently removed by depletion with
magnetic beads. Remaining cells were stained with goat antimouse
F(ab)2-FITC (Zymed, San Francisco, CA), CD11c PE and CD4 Tricolor and
the
CD11cCD4+CD3CD14CD19CD56
cells were sorted using a FACStar plus.
Antibodies.
FITC-labeled antibodies specific for CD3, CD5, CD7, CD8, CD10, CD14,
CD19, CD33, CD40, CD45RA, CD56, and CD69 were purchased from Becton
Dickinson. Anti-CD1a FITC was obtained from Serotec (Kidlington,
Oxford, UK), anti-CD33 FITC from Immunotech (Marseille, France),
anti-CD86 FITC from Instruchemie (Hilversum, The Netherlands), and
anti-CD40 FITC from Pharmingen (San Diego, CA). PE-labeled antibodies
against CD2, CD4, CD13, CD20, CD80, and HLA-DR were purchased from
Becton and Dickinson. Anti-CD1a PE, anti-CD54 PE, anti-CD83 PE, and
anti-T-cell receptor (TCR)  PE were purchased from
Coulter/Immunotech (Luminy, France), anti-CD11c PE from Biosource International (Nivelle, Belgium), and anti-IL-3R PE from Pharmingen. Anti-CD4tricolor was obtained from DAKO A/S (Glostrup, Denmark) and
anti-TCR Tricolor from Immunotech. The nonconjugated antibody against CD1a was purchased from Becton and Dickinson. Anti-CD45RA (GI-15) was a gift of Bristol-Meyers Squibb (Seattle, WA) and anti-CD86
(IT2.2) was kindly provided by M. Azuma (Juntendo University, Tokyo, Japan).
Immunohistochemical analysis.
Single staining was performed as described previously using a standard
immunoperoxidase method.13 Briefly, cryostat fragments of
thymic tissue were cut in 4-µm sections, air-dried overnight, and
fixed in acetone for 10 minutes at room temperature. The slides were
first incubated with 10% (vol/vol) normal rabbit serum (CLB), then
with optimal dilutions of primary MoAb (in phosphate-buffered saline
[PBS] containing 1% (wt/vol) bovine serum albumin [BSA] [PBS/BSA]) for 30 minutes at room temperature, followed by incubation with biotinylated goat antimouse IgG (DAKO A/S). After incubation with
streptavidin/biotin-conjugated peroxidase complex (ABC-protocol, DAKO),
the bound peroxidase was developed with 3-amino-9-ethylcarbazole (Sigma
Chemical Co, St Louis, MO), 0.4 mg/mL in 0.1 mol/L sodium acetate
buffer, pH 5.0. Between incubation steps, the sections were extensively
rinsed in PBS/BSA. The sections were counterstained with hematoxylin
and mounted. Within each staining procedure, negative control
antibodies were included.
For double-staining, acetone-fixed cryostat sections were incubated
subsequently with primary MoAb, biotinylated rabbit antimouse IgG and
streptavidin/biotin-conjugated peroxidase complex. After incubation
with normal mouse serum (CLB), sections were stained with either
PE-labeled or FITC-labeled secondary mouse MoAb. PE staining was
visualized by incubation with rabbit anti-PE (Biogenesis, Poole,
England, UK) followed by goat antirabbit-complexed to alkaline phosphatase (Immunotech) and FITC staining with sheep
anti-FITC-complexed to alkaline phosphatase (Boehringer Mannheim,
Almere, The Netherlands). Color development for alkaline phosphatase
and peroxidase was performed by incubations with naphtol AS-MX
phosphate (0.4 mg/mL) plus Fast blue BB base (0.6 mg/mL, Sigma, in 0.1 mol/L Tris-HCl, pH 8.5) and for peroxidase by incubation with and
3-amino-9-ethylcarbazole (0.4 mg/mL in 0.1 mol/L sodium acetate buffer,
pH 5.0).
For triple-staining, acetone fixed cryostat sections were incubated
subsequently with primary mouse antihuman MoAb, biotinylated rabbit
antimouse IgG, Cy5-conjugated streptavidin (Jackson Immunoresearch Laboratories, Inc, Palo Alto, CA), normal mouse serum, secondary PE-labeled MoAb, rabbit anti-PE (Biogenesis), Cy3-conjugated goat antirabbit (Jackson Immunoresearch Laboratories), normal mouse serum
and FITC-labeled tertiary MoAb. For each fluorochrome label, negative
control antibodies were included.
Confocal laser scanning microscope analysis.
Confocal fluorescence images were obtained on a Leica TCS NT (Leica
Microsystems, Heidelberg, Germany) confocal system, equipped with an
Ar/Kr laser. Images were taken using a 40x 1.25 NA objective. Possible
cross-talk between FITC, Cy3, and Cy5, which could give rise to
false-positive colocalization of different signals, was avoided by
careful selection of the imaging conditions. The standard FITC/Cy3/Cy5
filter combination and Kalman averaging was used. Color
photomicrographs were taken from electronic overlays. Processing of
images for presentation was performed on a PC using the software packages Photoshop (Adobe Systems Inc, Mountain View, CA) and Freelance
Graphics (Lotus Development Corp, Cambridge, MA).
Generation of DC in IL-3- and CD40L-supported cultures of thymic DC
precursors.
CD1aCD3CD4+CD8
thymocytes were cultured in a 96-well flat bottomed plate in the
presence of 10 ng/mL IL-3 with or without 104 CD40L
transfected mouse fibroblasts (kindly provided by J. Banchereau, Schering Plough, Dardilly, France) preirradiated with 104
rad. Culture medium consisted of Yssel's medium14 with 5%
normal human serum.
Staining of freshly isolated and IL-3-cultured thymic DC precursors
on slides.
Cytocentrifuge preparations of freshly isolated
CD1aCD3CD4+CD8
thymocytes were fixed with acetone, incubated in rabbit antihuman HLA-DR antibody (1/500 serum dilution, obtained from J. Neefjes, Netherlands Cancer Institute, Amsterdam, The Netherlands) for 30 minutes at room temperature, washed in PBS/BSA, followed by incubation
in Cy3-conjugated goat antirabbit antibody for 30 minutes at room
temperature. Clusters of cells that were cultured in the presence of
IL-3 for 5 days were resuspended in medium and layered on
3-amino-propyltriethoxysilane-coated glass slides. After 3 minutes at
37°C, adhering cells were fixed in Cellfix solution (1/10 diluted
solution of buffered 10% formalin/1% sodium azide) (Becton Dickinson)
for 30 minutes at room temperature. Upon subsequent incubations in
PBS/BSA for 5 minutes and in PBS/0.25% saponin for 30 minutes, cells
were stained for HLA-DR and confocal fluorescence images were obtained
as described above.
Hybrid human/mouse fetal thymic organ cultures.
The in vitro development of human T cells was studied using the hybrid
human/mouse fetal thymic organ culture (FTOC).9 Fetal
thymuses were dissected from embryos of recombination activating gene-1
(RAG-1)-deficient mice on day 15 to 16 of gestation and precultured
for 5 days in the presence of 1.35 mmol/L 2-deoxyguanosine (Sigma) to
remove endogenous thymocytes. Next, the thymic lobes were cocultured
for 2 days in hanging drops in wells of a Terasaki plate with human
progenitor cells and transferred to nucleopore filters, which were
layered over gelfoam rafts in 6-well plates (Costar, Badhoevedorp,
Netherlands). The lobes populated with human cells were cultured for
the indicated number of days in Yssel's medium supplemented with
2% normal human serum and 5% fetal calf serum. To analyze
differentiation of human cells, the mouse thymuses were dispersed into
single cell suspensions and stained with MoAbs specific for human cell
surface antigens.
Reverse transcriptase-polymerase chain reaction (RT-PCR) assays.
RNA was isolated from fluorescence-activated cell sorting (FACS)-sorted
or cultured postnatal thymocytes and tonsil cells using TRIzol reagent
(GIBCO, Paisley, Scotland) according to the manufacturer's
instructions and reverse transcribed using a poly-dT15 oligonucleotide (Promega, Madison, WI) and 400 U of
Moloney murine leukemia virus (M-MuLV) reverse
transcriptase (GIBCO) at 42°C for 1 hour. PCR assays were performed
in 50 µL reaction volumes using 1 µL of cDNA template, 2 mmol/L
MgCl2, 0.25 mmol/L of each dNTP, 1 µmol/L of
each primer and 3 U Taq-polymerase (GIBCO) in 1× buffer (10 mmol/L
TRIS-HCl, pH 8.5, 50 mmol/L KCl). Reaction conditions were a 5-minute
denaturation step at 94°C followed by 30 cycles of 1 minute at
94°C, 1 minute at 65°C, and 2 minutes at 72°C. PCR products
were separated on 1.2% agarose gels, stained with ethydium bromide,
and analyzed by videodensitrometry using the Eagle Eye still video
system and Eagle Sight Software (Stratagene, La Jolla, CA).
The hydroxyphosphoribosyl transferase (HRPT), RAG-1, and
pT primers that were used are:
HPRT sense: 5'-TATGGACAGGACTGAACGTCTTGC-3', HPRT antisense:
5'-GACACAAACATGATTCAAATCCCTGA-3', RAG-1 sense:
5'-GAACACACTTTGCCTTCTCTTTGG-3', RAG-1 antisense:
5'-CGCTTTGCCTCTTGCTTTCTCGTT-3', pT sense:
5'-GTCCAGCCCTACCCACAGGTGT-3', pT antisense:
5'-CGGGAATTCGACGTCCCTGGCTGTAGAAGCCTCTC-3'.
Semiquantitative RT-PCR (semi-Q RT-PCR).
To compare the amount of pT mRNAs expressed in different samples,
expression was related to the level of expression of the housekeeping
enzyme HPRT. Standard curves for HPRT and pT were established by
simultaneous amplification of serial dilutions of cDNA prepared from
RNA isolated from total thymus. Total thymocytes contain a high
concentration of the target sequence being amplified to ensure that
quantitative values fell within the linear range of the standard curve.
PCR was performed in a total volume of 50 µL consisting of 1 µmol/L
of each primer set, 200 µmol/L each dNTP (Pharmacia Biotech, Uppsala,
Sweden), 2.5 mmol/L MgCl2, 1x PCR buffer, 1 U Taq DNA
polymerase (GIBCO-BRL, Gaithersburg, MD), and 10 µL of
the sample cDNA. Samples were covered with 50 µL paraffin oil and
heated to 94°C for 5 minutes followed by amplification for 30 cycles of 1 minute 94°C, 1 minute 65°C (HPRT, RAG-1), or 1 minute 59°C (pT), and 2 minutes 72°C. After the last cycle, a final extension step at 72°C for 10 minutes was performed. A total of 10 µL of each PCR reaction was dotblotted in duplicate onto
a nylon filter (Hybond N+, Amersham International plc,
Slough, UK). Filters were prehybridized at 60°C for at least 1 hour
(6x SSC, 0.5% sodium dodecyl sulfate [SDS], 5x Denhardt's, 100 mg
herring sperm DNA/L) and hybridized overnight with an oligoprobe
specifically recognizing the HPRT or pT PCR product internal to the
PCR primers. The sequences of the probes were:
pT probe: 5'-CTGCCTTCTGAGGAGCTGGCAT-3'. HPRT probe:
5'-GTCCCCTGTTGACTGGTCATTACAAT-3'.
Oligoprobes were 32-P endlabeled according to the
manufacturer's recommendations (Boehringer Mannheim). To remove any
nonspecifically bound probe, the filters were washed with excess amount
of 2x SSC; 0.1% SDS at 55°C for 30 minutes. Measurement of the
cpm/dot values was performed with a phosphoimager (Fujix Bas 2000, Fuji). Standard curves were calculated using the Softmax
program (Molecular Dynamics, Sunnyvale, CA), in which the
first point of the curve, containing the highest amount of cDNA, was
set arbitrarily to 1,000 U. Using the same Softmax program, also the
values of the samples were calculated in units. Finally the ratios of
(pT)/(HPRT) was calculated to compare the expression of the above
mRNA in the different samples.
NK cell assays.
Thymocytes were cultured in 96-well U bottom plates in Yssel's medium
supplemented with 5% normal human serum in the presence of 25 U/mL
Flt-3 ligand (Flt-3L from Peprotech, Rocky Hill, NJ), 10 ng/mL IL-7
(Peprotech,) and 10 ng/mL IL-15 (Peprotech) or in the presence of 10 ng/mL IL-7, 20 ng/mL stem cell factor (SCF, Amgen, Thousand Oaks, CA)
and 50 U/mL IL-2 (EuroCetus, Amsterdam, The Netherlands).
| |
RESULTS |
Identification of a thymic
CD3CD4+CD8
population, which is similar to tonsillar plasmacytoid T cells.
Sotzic et al11 have described a CD4+ thymic
population that lacks CD1a. Upon overnight culture, these cells
acquired DC characteristics. It was unclear whether these cells
represent mature DC or DC precursors. In this study, we isolated
CD1aCD3CD4+CD8
cells from the thymus and examined their cell surface phenotype extensively by flow cytometry. As illustrated in
Fig 1A, these cells lack expression of
markers for NK (CD56), B cells (CD10, CD19, and CD20), and monocytes
(CD14), which is consistent with previous observations.11
Further analysis showed that
CD1aCD3CD4+CD8
thymocytes express CD44, CD45RA, HLA-DR, and CD54, but not CD13, CD33,
CD80, and CD86 (Fig 1A). This phenotype is similar to that of a
committed DC precursor in the tonsil, previously referred to as
plasmacytoid T cells.12 However, differences were observed, as in contrast to plasmacytoid T cell, all thymic
CD1aCD3 CD4+CD8
cells expressed the T/NK cell markers CD2 and CD7 and most of these
cells expressed CD5 (Fig 1A). These markers are also expressed at
specific stages of NK development. To exclude that expression of these
markers was due to passive absorption of CD2, CD5, and CD7 shed from
thymocytes, we reanalyzed expression of CD2, CD5, and CD7 after a 5-day
culture in IL-3. Under these conditions, no contaminating thymocytes
survive. Figure 1B shows that cultured cells still express significant
levels of CD2, CD5, and CD7, although the level of expression of CD7 is
much lower than on freshly isolated cells.
View larger version (49K):
[in this window]
[in a new window]
| Fig 1.
FACS analysis of thymic DC precursors. (A) Thymocytes
sorted on basis of a
CD1aCD3CD4+CD8
phenotype were analyzed for the expression of a number of lymphoid,
myeloid, costimulatory, and adhesion markers. (B) Expression of CD2,
CD5, and CD7 on
CD1aCD3CD4+CD8
cells cultured for 5 days in IL-3.
|
|
CD1aCD3CD4+CD8
thymocytes develop to mature dendritic cells upon stimulation with IL-3
together with CD40L.
Tonsillar DC precursors can differentiate into DC when cultured in the
presence of IL-3 and CD40L expressing fibroblasts.12 Given
the similarity of the
CD1aCD3CD4+CD8
thymocytes with the DC precursors found in the tonsil, we examined the
response of the thymic
CD1aCD3CD4+CD8
thymocytes to the same culture conditions. After a few hours, these
cultures contained large cell clusters. IL-3 together with CD40L
induced the strongest activation of DC cells. In the first 5 days, the
cells activated with IL-3 and CD40L did not expand significantly, but
in the period of 5 to 10 days, the cells expanded 2-fold to 3-fold.
IL-3 alone was sufficient for generation of DC, but was less efficient
than in combination with CD40L. After stimulation with IL-3, the DC
precursor cells with rounded cell morphology
(Fig 2A) developed into a
mature DC with a typical veiled dendritic like morphology, exhibiting
enhanced expression of HLA-DR (Fig 2B). Furthermore, stimulation of
CD1aCD3CD4+CD8
thymocytes with IL-3 and CD40L resulted in the upregulation of expression of the costimulatory molecules CD80 and CD86 and the DC-specific marker CD83 (Fig 2C). The DC obtained after culture with
IL-3 and CD40L were potent activators of proliferation of allogeneic T
cells in mixed cell cultures (data not shown). These data demonstrate
that the thymic
CD1aCD3CD4+CD8
cells have DC precursor activities similar to those of the previously described committed DC precursors in the tonsil. Consistent with the
responsiveness of the thymic DC precursors to IL-3, these cells express
IL-3R (Fig 2D). In contrast, these cells fail to express the
IL-7R chain and were unable to respond to IL-7. As a comparison, we
studied the expression of both molecules on
CD1a+CD3CD4+CD8
immature single positive (CD4 ISP) thymocytes. As indicated, this
latter population did not express IL-3R, but displayed low levels of
IL-7R (Fig 2D). CD4 ISP cells fail to respond to IL-3 and died
within 2 days of culture, consistent with the lack of IL-3R on these
cells.
View larger version (98K):
[in this window]
[in a new window]
| Fig 2.
Morphology and phenotype of thymic DC precursors freshly
isolated and after in vitro stimulation with IL-3. For confocal laser
scanning microscope analysis,
CD1aCD3CD4+CD8
thymocytes were sorted and either directly cytocentrifuge preparations
were made or cells were cultured for 5 days in the presence of IL-3,
resuspended and layered on precoated glass slides. Both fresh and
cultured cells were stained for HLA-DR. For FACS analysis,
CD1aCD3CD4+CD8
thymocytes were cultured for 5 days in the presence of IL-3 and
CD40L+ mouse fibroblasts. (A) Freshly isolated DC
precursors show a rounded morphology and weak staining for HLA-DR. (B)
Activated DCs upon culture in IL-3 acquire a dendritic morphology and
more intense staining for HLA-DR (bar, 5 µm). (C) IL-3- and
CD40L-activated DCs display a phenotype specific for mature DC. (D) The
expression of IL-3R and IL-7R on thymic DC precursors compared
with CD4 ISP pre-T cells. Solid lines represent staining with specific
antibodies as compared with staining with isotype-matched controls
indicated with dotted lines.
|
|
CD1aCD3CD4+CD8
thymic DC precursors are predominantly located in the medulla.
As shown in Fig 2D, thymic DC precursors express high levels of
IL-3R. To explore whether this feature is unique for these DC, we
analyzed IL-3Rhigh cells in a sample of total
unseparated thymocytes. Total thymocytes were stained with antibodies
against CD4 and IL-3R and either CD1a or CD45RA. Electronically
gated CD4+ IL-3Rhigh cells were analyzed for
CD1a and CD45RA expression. As shown in Fig
3, the great majority of the CD4+ IL-3Rhigh
expressed CD45RA and lacked CD1a strongly suggesting that the majority
of cells that express high levels of IL-3R are identical to the
precursors characterized in the preceding experiments (Figs 1 and 2).
In addition, very small populations of CD4
IL-3Rhigh and CD1a+CD4+
IL-3Rhigh were detectable. It is possible that these
cells represent intermediate stages of DC development in the thymus.
Unfortunately this point could not be unambiguously checked. Because of
the small sizes of these populations, we were unable to purify them
sufficiently to evaluate their DC precursor activities.
View larger version (27K):
[in this window]
[in a new window]
| Fig 3.
The majority of thymocytes with a high level of IL-3R
are thymic DC precursors. Total thymocytes were stained for three-color
FACS analysis with anti-IL-3RPE, anti-CD4 Tricolor and either
anti-CD1 FITC or anti-CD45RA FITC. The histograms show the staining of
CD1a and CD45RA of CD4+ IL-3Rhigh cells as
gated in the dot plot (0.1% of total thymocytes).
|
|
We made use of the elevated expression of IL-3R by the thymic DC
precursors to study their localization in the thymus using immunohistochemistry on tissue sections.
Figure 4A shows that IL-3Rhigh cells are predominantly located in the
medulla, whereas a few cells are also present in the corticomedullary
border and the cortex. In line with the phenotype observed for freshly
isolated DC precursors, the majority of IL-3Rhigh
thymocytes in the medulla coexpressed CD45RA (not shown) and lacked
CD1a (Fig 4B). Many thymic pre-DC were in close
contact with CD1a+ thymocytes (Fig 4B, inset). Three-color
immunofluorescence analyses of tissue sections furthermore demonstrated
that the medullary IL-3Rhigh thymocytes showed virtually
no costaining with the costimulatory molecule CD86
(Fig 5A). In addition, there was no
distinct costaining of IL-3Rhigh cells with CD83, which
is a marker specific for mature DC (Fig 5A and E). However, the medulla
does contain a population of CD83+ cells, which lack
significant IL-3R expression. Many of these mature DC coexpress CD86
(Fig 5A).
View larger version (141K):
[in this window]
[in a new window]
| Fig 4.
Thymic localization of DC precursors. (A) Thymic tissue
sections were incubated with anti-IL3R MoAb and counterstained with
hematoxylin. IL-3Rhigh cells (red), which comprise
DC precursors, are predominantly located in the medulla and a few
are scattered through the cortex. (B) Thymic tissue sections were
double-stained for IL-3R and CD1a. IL-3Rhigh cells
(blue) are often found adjacent to CD1a+ thymocytes (red)
(see inset). Original magnification (A) ×200, (B) ×400.
|
|
View larger version (75K):
[in this window]
[in a new window]
| Fig 5.
The medulla contains both DC precursors and mature DC.
Thymic tissue sections were triple-stained with anti-IL-3R (red),
anti-CD83 (green), and anti-CD86 (blue). Patterns of the individual
colors, which combined are shown in (A), are depicted separately in (B)
(anti-CD83), (C) (anti-IL-3R ), and (D) (anti-CD86). As a comparison
in (E), a control staining of anti-IL-3R (red), anti-CD83 (green),
and control IgG (blue) is included. The round structure on the left
side represents a Hassall's corpuscle, which indicates the thymic
medulla. Note in (A) the red stained pre-DC versus the blue/green
stained mature DC. Original magnification ×400.
|
|
CD1aCD3CD4+CD8
thymic DC precursors express high levels of mRNA for the pre-TCR
chain.
The phenotype of thymic DC precursors does not only resemble that of DC
precursors in the tonsil, but also that of a population of cells
present in adult peripheral blood, which has been shown to contain
committed T-cell precursors.15 This latter
CD1aCD3CD4+CD8CD14
population of medium sized peripheral blood mononuclear cell (PBMC) has
been shown to give rise to mature CD4+ SP
TCR+ T cells in a thymus-independent fashion. TCR
DJ, but no VDJ rearrangements, were detected in these
cells.15 Furthermore these cells were found to express mRNA
for pT and RAG-1. Although not shown, the peripheral blood
CD1aCD3CD4+CD8CD14
population contains also cells that express high levels of IL-3R, able to differentiate into mature DC in the presence of IL-3 and CD40L.
The high level of expression of pT mRNA in
CD1aCD3CD4+CD8CD14
PBMC15 prompted us to examine pT mRNA levels in thymic
DC precursors. As controls, we included
CD1aCD3+CD4+ mature
thymocytes that should lack pT16 and
CD1a+CD3CD4+CD8
ISP pre-T cells17-19 expected to express pT
mRNA.16 Figure 6 demonstrates
that not only committed pre-T cells express pT mRNA, but also the
thymic DC precursors. The ratio of the pT and HPRT signal
intensities in the DC precursors is even higher than in the
CD4+ pre-T cells, suggesting that the detected pT mRNA
in the pre-DC is not due to contamination with CD4 ISP pre-T cells. We
also detected high levels of pT mRNA in cells that were isolated
from CD8-depleted thymocytes on the basis of expression of CD4,
IL-3Rhigh and CD45RA. These pT mRNA levels were
higher that those of
CD45RACD4+IL-3R
cells obtained from the same sort (results not shown). Although the
level of expression was decreased, pT mRNA was still observed in the
thymic DC after 2 days of culture with IL-3. This observation confirms
the notion that pT expression in these cells is not due to
contamination with CD4 ISP pre-T cells, as these latter cells are
unable to survive for 2 days with IL-3 (Fig 6). After 7 days of
culture, the mature DC did not express pT mRNA anymore, suggesting
that concomitant with acquisition of a mature phenotype, pT is
downregulated. As expected, the
CD1aCD3+CD4+ SP mature
thymocytes lacked pT mRNA. The CD4 ISP thymocytes displayed RAG-1
mRNA expression, in contrast to the mature CD4 SP thymocytes and the
thymic DC precursors. To investigate whether pT mRNA expression is
specific for thymic DC precursors, we examined pT mRNA expression in
lineage markers negative
CD11cCD3CD4+ DC
precursors from the tonsil. Figure 6 shows that pT mRNA is present
in these DC precursors as well.
View larger version (28K):
[in this window]
[in a new window]
| Fig 6.
Thymic and tonsillar DC precursors express mRNA for
pT. Thymic DC precursors, CD4 ISP, mature CD4 SP thymocytes, and
tonsillar DC precursors were analyzed for expression of mRNA for HPRT,
pT, and RAG-1. pT mRNA expression was also determined for thymic
DC precursors cultured for 2 and 7 days with IL-3.
|
|
The earliest precursors in the thymus express CD34 and lack CD1a. This
population contains T, NK, and DC precursor activities.8,9 Upon upregulation of CD1a, the DC precursor activity is lost and the NK
precursor activity is strongly diminished.20 The
CD34+CD1a+ cells differentiate into CD4 ISP
pre-T cells. To obtain insight into the levels of pT in different
subsets of early precursors in the thymus, we performed a
semiquantitative PCR with CD34+CD1a,
CD34+CD1a+, and
CD3CD4+CD8 cells from
the same thymic sample. These latter cells were sorted on the basis of
CD4 expression and absence of CD3 and CD8 and therefore include pre-T
cells and committed pre-DC. The pre-DC constitute 10% of the sorted
CD3-CD4+CD8 population. As was found
previously for fetal thymic CD34+CD1a
cells,21 the postnatal
CD34+CD1a cells express low levels of
pT mRNA (Fig 7). Around 10-fold higher
levels of pT mRNA were observed in
CD34+CD1a+ cells, which increased a further
1.5-fold in CD3-CD4+CD8 cells (Fig 7).
It is feasible that the CD4+ pre-DC are responsible for
this increase. These results shown in Figs 6 and 7 indicate that pT
is present in uncommitted thymic precursors, but at levels lower than
those of the committed IL-3R+ DC precursors.
View larger version (75K):
[in this window]
[in a new window]
| Fig 7.
Expression levels pre-T mRNAs in subsets of thymic
precursors. Total RNA was isolated from sorted
CD34+CD1a,
CD34+CD1a+, and CD4 ISP human thymic
subpopulations (> 99% pure upon reanalysis) and analyzed for the
levels of pT mRNA expression by semiquantitative RT-PCR as described
in Materials and Methods (A). mRNA levels were compared with HPRT
expression and calculated in arbitrary units. Standard curves were
established by simultaneous amplification of 2-fold dilutions of cDNA
prepared from RNA isolated from total thymus. PCR product of standard
curves and samples were dot-blotted in duplicate onto nylon filters and
hybridized with end-labeled oligo nucleotides shown on the right-hand
site of the figure. (B) Measurement of the cpm/dot values was performed
with a phosphoimager. Standard curves were calculated in which the
first point of the curve, containing the highest amount of cDNA, was
set arbitrarily to 1,000 U. Values of the samples were calculated in
units. The ratios of (pT)/(HPRT) were determined and visualized in
the bar graph.
|
|
CD1aCD3CD4+CD8
thymocytes are unable to develop into T cells or NK cells.
The cell surface expression of CD2, CD5, and CD7 and the presence of
expression of pT mRNA in the thymic
CD1aCD3CD4+CD8
pre-DC population raised the possibility that these cells may have
T-cell precursor activity. Therefore, we studied the potential of these
cells to develop to T cells in fetal thymic organ culture. As a
positive control, we included the CD4 ISP thymocytes. As expected,
these CD4 ISP thymocytes developed through a DP stage into mature
TCR+ T cells (Fig 8).
This population gave, in addition, rise to a small percentage of
TCR+ T cells. Very few cells could be recovered from
the FTOC with the pre-DC. Those cells did not express CD8, TCR,
or TCR, while a proportion still expressed CD4. Presumably these
CD3CD4+CD8 cells are
surviving pre-DC. These data indicate that
CD1aCD3CD4+CD8
thymocytes were unable to generate T cells in an FTOC.
View larger version (33K):
[in this window]
[in a new window]
| Fig 8.
CD1aCD3CD4+CD8
thymocytes do not contain T-cell precursors. The ability of
CD1aCD3CD4+CD8
thymocytes to give rise to T cells was tested in a human/mouse FTOC. As
a comparison, the developmental capacity of
CD1a+CD4+ ISP thymocytes was examined.
|
|
Because the
CD1aCD3CD4+CD8CD14
peripheral blood population has been shown to generate T cells when
directly stimulated with phytohemagglutinin (PHA), IL-2, IL-7, and
allogeneic feeder cells,15 this same stimulation regimen
was applied to the
CD1aCD4+CD3CD8
thymocytes. Also under these conditions, no T-cell development was
observed (data not shown). Taken together, these results therefore suggest that the
CD1aCD3CD4+CD8
thymocytes do not contain T-cell precursors. To investigate NK precursor activity, we cultured the thymic
CD1aCD3CD4+CD8
cells in either a mixture of IL-2, IL-7, and SCF or a mixture of IL-7,
IL-15, and Flt-3L.22 While both mixtures simulated differentiation of NK cells from CD34+ thymocytes, they
failed to induce NK cell development from the thymic
CD1aCD3CD4+CD8
cells (results not shown).
| |
DISCUSSION |
Here we have characterized CD4+ thymic precursors committed
to the DC lineage. Cells within this population lack CD1a, express high
levels of CD45RA and the IL-3R chain, and are predominantly located
in the thymic medulla. They develop into mature DC in vitro upon
stimulation with IL-3 or IL-3 and CD40, but are unable to develop into
T and NK cells in an FTOC or in mixtures of either IL-7, IL-15, and
Flt-3L or IL-2, IL-7, and SCF. These committed DC precursors share many
characteristics with pre-T cells. Both cell populations express CD2,
CD5, and CD7. These antigens could be detected on DC precursors
cultured in IL-3, indicating that expression of CD2, CD5, and CD7 was
not due to absorption from T-lineage cells within the thymus. More
strikingly, we detected pT mRNA in the committed DC precursors. This
was unexpected because pT is considered to be specific for the
T-cell lineage. Because we determined expression of pT mRNA at a
population level, we cannot completely rule out that pT is expressed
in cells within the population that are not committed DC precursors.
However, we observed that high levels of pT were also present in
cells that were sorted on the basis of IL-3R expression, that
CD1aCD4+ pre-DC contain more pT mRNA
than CD1a+CD4+ pre-T cells, and that pT mRNA
could still be detected in DC precursors cultured for 2 days in IL-3.
These observations together support the interpretation that the
committed DC precursors do express pT mRNA. The
IL-3R+ DC precursors show also similarities with NK
cells, as both cell types express CD2, CD5, and CD7. Whether committed
NK precursors express pT mRNA is not known, but it is interesting to
note that pT mRNA was detected in T/NK precursor cells from murine
fetal blood.23
The similarity in phenotype of pre-T cells, pre-NK cells, and pre-DC
strongly suggest a common origin of these cells. The observation that
pT transcripts, detected in both committed pre-T and pre-DC, are
also expressed in CD34+CD1a cells that
have T, DC, and NK precursor activities8,9 is consistent
with this notion. Such a developmental relationship of T cells and DC
has been inferred from the seminal studies of Shortman et
al,4 who demonstrated that mouse intrathymic DC and T cells
are derived from a common
CD4lowCD44+CD25 thymic
precursor. This latter population can also produce B cells and NK
cells24 and therefore may contain common lymphoid
precursors (CLP).25 Because the mouse thymic
DC originate from cells, which lack myeloid precursor activity, they
were called lymphoid DC to distinguish them from DC derived through the
myeloid lineage. Further studies in the mouse have made clear that
lymphoid-derived CD8+ DC are also present in the spleen
and the circulation.26,27 The peripheral lymphoid dendritic
cells may well arise from a CLP population that has been found in bone
marrow and which may be the source of cells that colonize the
thymus.25 In humans, there is also evidence for a lymphoid
origin of a subset of DC. Galy et al28 have identified a
CLP in the bone marrow, which can generate T, B, and NK and also DC,
but not myeloid cells. We have found an almost identical precursor in
the human thymus. Like the CLP, these cells express CD10, CD34 and
CD45RA lack CD1a and can, next to T cells, develop into NK or DC
dependent on the cytokines added to these cells.9 It is
possible that the thymic committed pre-DC, identified here, are the
progeny of the
CD34+CD1aCD10+CD45RA+
thymic precursors and therefore may represent human lymphoid DC. It is,
however, important to note that this hypothesis has yet to be directly
verified. Not only the developmental origin of the
IL-3R+ DC precursors has yet to be determined, but also
their final destination. Our data indicate that, apart from committed
pre-DC, the medulla contains mature CD83+CD86+
DC, which lack IL-3R. Whether the IL-3R+ pre-DC give
rise to these medullary mature DC in vivo is unknown. A detailed
comparison between the mature CD83+, the
IL-3R+ pre-DC, and their progeny is necessary to resolve
this point.
The finding that DC precursors (plasmacytoid T cells) with similar
features as thymic DC precursors are present in the
tonsil12 indicates that this cell type is not confined to
the thymus. Both cell populations express high levels of CD45RA,
IL-3R, and pT mRNA, but lack CD1a and express only very low
levels of CD11c. Moreover, these cells respond to IL-3 and the
combination of IL-3 and CD40L in an identical way. There appears to be
a similar population in peripheral blood. Recently we identified a
CD3CD4+CD8CD14
population in the peripheral blood of adults.15 Cells in
this population expressed high levels of pT mRNA. However, in
contrast to the pre-DC in the thymus, they also expressed RAG-1 mRNA
and demonstrated T-cell precursor activity. Confirming results of other
groups, we have also found that
CD3CD4+CD8CD14
peripheral blood cells have DC precursor activities (L. Bruno, P. Res,
and H. Spits, unpublished findings). In addition, we observed that this
population contains cells with a high level of IL-3R and, although
this was not directly tested, it is likely that these
IL-3R+ cells also express pT mRNA. It is clear that
the DC precursor found in the thymus is not unique for this organ. As
discussed above, we speculate that the thymic IL-3R+ DC
precursors originate from an intrathymic CD34+ progenitor,
although this has yet to be directly proven. An interesting question is
whether the pT+ DC precursors found in peripheral blood
and tonsil are derived from the common lymphoid/DC precursors present
in bone marrow or from the CD34+ thymocytes. In the latter
case, pT mRNA expression in lymphoid DC could be a thymus-induced event.
| |
ACKNOWLEDGMENT |
The authors thank Lauran Oomen and Ilja van de Pavert for help with
confocal microscopy analysis and Trees Dellemijn and Natascha Verra for
preparation and antibody labeling of tissue sections. Bianca Blom is
acknowledged for her help with the semiquantitative PCR.
| |
FOOTNOTES |
Submitted February 16, 1999; accepted June 9, 1999.
Supported by Grant No. NKI 95-960 from the Dutch Cancer Society.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests Hergen Spits, MD, Division of
Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX
Amsterdam, The Netherlands; e-mail: hergen{at}nki.nl.
| |
REFERENCES |
1.
Banchereau J, Steinman RM:
Dendritic cells and the control of immunity.
Nature
392:245, 1998[Medline]
[Order article via Infotrieve]
2.
Steinman RM, Pack M, Inaba K:
Dendritic cells in the T-cell areas of lymphoid organs.
Immunol Rev
156:25, 1997[Medline]
[Order article via Infotrieve]
3.
Sprent J, Webb SR:
Intrathymic and extrathymic clonal deletion of T cells.
Curr Opin Immunol
7:196, 1995[Medline]
[Order article via Infotrieve]
4.
Ardavin C, Wu L, Shortman K:
Thymic dendritic cells and T cells develop stimultaneously in the thymus from a common precursor population.
Nature
362:761, 1993[Medline]
[Order article via Infotrieve]
5.
Kronin V, Winkel K, Suss G, Kelso A, Heath W, Kirberg J, Von Boehmer H, Shortman K:
A subclass of dendritic cells regulates the response of naive CD8 T cells by limiting their IL-2 production.
J Immunol
157:3819, 1996[Abstract]
6.
Suss G, Shortman K:
A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis.
J Exp Med
183:1789, 1996[Abstract/Free Full Text]
7.
Wu L, Li CL, Shortman K:
Thymic dendritic cell precursors: Relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny.
J Exp Med
184:903, 1996[Abstract/Free Full Text]
8.
Marquez C, Trigueros C, Fernandez E, Toribio ML:
The development of T and non-T cell lineages from CD34+ human thymic precursors can be traced by the differential expression of CD44.
J Exp Med
181:475, 1995[Abstract/Free Full Text]
9.
Res P, Martínez Cáceres E, Jaleco AC, Noteboom E, Weijer K, Spits H:
CD34+CD38dim cells in the human thymus can differentiate into T, natural killer and dendritic cells but are distinct from stem cells.
Blood
87:5196, 1996[Abstract/Free Full Text]
10.
Schmitt C, Ktorza S, Sarun S, Blanc C, de Jong R, Debré P:
CD34 expressing human thymocyte precursors proliferate in response to IL-7 but have lost myeloid differentiation potential.
Blood
82:3675, 1993[Abstract/Free Full Text]
11.
Sotzik F, Rosenberg Y, Boyd AW, Honeyman M, Metcalf D, Scollay R, Wu L, Shortman K:
Assessment of CD4 expression by early T precursor cells and by dendritic cells in the human thymus.
J Immunol
152:3370, 1994[Abstract]
12.
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
185:1101, 1997[Abstract/Free Full Text]
13.
Vyth-Dreese F, Dellemijn TA, Majoor D, De Jong D:
Localization in situ of the co-stimulatory molecules B7.1, B7.2, CD40 and their ligands in normal human lymphoid tissue.
Eur J Immunol
25:3023, 1995[Medline]
[Order article via Infotrieve]
14.
Yssel H, De Vries JE, Koken M, Van Blitterswijk W, Spits H:
Serum-free medium for the generation and the propagation of functional human cytotoxic and helper T cell clones.
J Immunol Methods
72:219, 1984[Medline]
[Order article via Infotrieve]
15.
Bruno L, Res P, Dessing M, Cella M, Spits H:
Identification of a committed T cell precursor population in adult human peripheral blood.
J Exp Med
185:875, 1997[Abstract/Free Full Text]
16.
Del Porto P, Bruno L, Mattei MG, Von Boehmer H, Saint-Ruf C:
Cloning and comparative analysis of the human pre-T-cell receptor -chain gene.
Proc Natl Acad Sci USA
92:12105, 1995[Abstract/Free Full Text]
17.
Hori T, Cupp J, Wrighton N, Lee F, Spits H:
Identification of a novel human thymocyte subset with a phenotype of CD3, CD4+,CD8+: Possible progeny of the CD3,CD4,CD8 subset.
J Immunol
146:4078, 1991[Abstract]
18.
Kraft D L, Weissman IL, Waller EK:
Differentiation of CD348 human fetal thymocytes in vivo: Characterization of a CD34+8 intermediate.
J Exp Med
178:265, 1993[Abstract/Free Full Text]
19.
Galy A, Verma S, Barcena A, Spits H:
Precursors of CD3+CD4+CD8+ cells in the human thymus are defined by expression of CD34. Delineation of early events in human thymic development.
J Exp Med
178:391, 1993[Abstract/Free Full Text]
20.
Spits H, Blom B, Jaleco A, Weijer K, Verschuren MCM, van Dongen JJ, Heemskerk MH, Res PC:
Early stages in the development of human T, natural killer and thymic dendritic cells.
Immunol Rev
165:75, 1998[Medline]
[Order article via Infotrieve]
21.
Blom B, Res PC, Noteboom E, Weijer K, Spits H:
Prethymic CD34+ progenitors capable of development into T cells are not committed to the T cell lineage.
J Immunol
158:3571, 1997[Abstract]
22.
Jaleco AC, Blom B, Res P, Weijer K, Lanier LL, Phillips JH, Spits H:
Fetal liver contains committed NK progenitors, but is not a site for development of CD34+ cells into T cells.
J Immunol
159:694, 1997[Abstract]
23.
Carlyle JR, Zuniga-Pflücker J:
Requirement for the thymus in T lymphocyte lineage commitment.
Immunity
9:187, 1998[Medline]
[Order article via Infotrieve]
24.
Wu L, Antica M, Johnson GR, Scollay R, Shortman K:
Developmental potential of the earliest precursor cells from the adult mouse thymus.
J Exp Med
174:1617, 1991[Abstract/Free Full Text]
25.
Kondo M, Weissman IL, Akashi K:
Identification of clonogenic common lymphoid progenitors in mouse bone marrow.
Cell
91:661, 1997[Medline]
[Order article via Infotrieve]
26.
Vremec D, Zorbas M, Scollay R, Saunders DJ, Ardavin CF, Wu L, Shortman K:
The surface phenotype of dendritic cells purified from mouse hymus and spleen: Investigation of the CD8 expression by a subpopulation of dendritic cells.
J Exp Med
176:47, 1992[Abstract/Free Full Text]
27.
Vremec D, Shortman K:
Dendritic cell subtypes in mouse lymphoid organs: Cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes.
J Immunol
159:565, 1997[Abstract]
28.
Galy A, Travis M, Cen D, Chen B:
Human T, B, natural killer, and dendritic cells arise from a common bone marrow progenitor subset.
Immunity
3:459, 1995[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
T. Matsui, J. E. Connolly, M. Michnevitz, D. Chaussabel, C.-I Yu, C. Glaser, S. Tindle, M. Pypaert, H. Freitas, B. Piqueras, et al.
CD2 Distinguishes Two Subsets of Human Plasmacytoid Dendritic Cells with Distinct Phenotype and Functions
J. Immunol.,
June 1, 2009;
182(11):
6815 - 6823.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Ishikawa, H. Niiro, T. Iino, S. Yoshida, N. Saito, S. Onohara, T. Miyamoto, H. Minagawa, S.-i. Fujii, L. D. Shultz, et al.
The developmental program of human dendritic cells is operated independently of conventional myeloid and lymphoid pathways
Blood,
November 15, 2007;
110(10):
3591 - 3660.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Schmidlin, W. Dontje, F. Groot, S. J. Ligthart, A. D. Colantonio, M. E. Oud, E. J. Schilder-Tol, M. Spaargaren, H. Spits, C. H. Uittenbogaart, et al.
Stimulated plasmacytoid dendritic cells impair human T-cell development
Blood,
December 1, 2006;
108(12):
3792 - 3800.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Dontje, R. Schotte, T. Cupedo, M. Nagasawa, F. Scheeren, R. Gimeno, H. Spits, and B. Blom
Delta-like1-induced Notch1 signaling regulates the human plasmacytoid dendritic cell versus T-cell lineage decision through control of GATA-3 and Spi-B
Blood,
March 15, 2006;
107(6):
2446 - 2452.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. I. Slukvin, M. A. Vodyanik, J. A. Thomson, M. E. Gumenyuk, and K.-D. Choi
Directed Differentiation of Human Embryonic Stem Cells into Functional Dendritic Cells through the Myeloid Pathway.
J. Immunol.,
March 1, 2006;
176(5):
2924 - 2932.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G.-X. Yang, Z.-X. Lian, K. Kikuchi, Y. Moritoki, A. A. Ansari, Y.-J. Liu, S. Ikehara, and M. E. Gershwin
Plasmacytoid Dendritic Cells of Different Origins Have Distinct Characteristics and Function: Studies of Lymphoid Progenitors versus Myeloid Progenitors
J. Immunol.,
December 1, 2005;
175(11):
7281 - 7287.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. J. Loza, P. Luppi, K. Kiefer, E. S. Martin, J. L. Szczytkowski, and B. Perussia
Human peripheral CD2-/lo T cells: an extrathymic population of early differentiated, developing T cells
Int. Immunol.,
September 1, 2005;
17(9):
1213 - 1225.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Drenou, L. Amiot, N. Setterblad, S. Taque, V. Guilloux, D. Charron, R. Fauchet, and N. Mooney
MHC class II signaling function is regulated during maturation of plasmacytoid dendritic cells
J. Leukoc. Biol.,
April 1, 2005;
77(4):
560 - 567.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Garnache-Ottou, L. Chaperot, S. Biichle, C. Ferrand, J.-P. Remy-Martin, E. Deconinck, P. D. de Tailly, B. Bulabois, J. Poulet, E. Kuhlein, et al.
Expression of the myeloid-associated marker CD33 is not an exclusive factor for leukemic plasmacytoid dendritic cells
Blood,
February 1, 2005;
105(3):
1256 - 1264.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. B. Gurney, A. D. Colantonio, B. Blom, H. Spits, and C. H. Uittenbogaart
Endogenous IFN-{alpha} Production by Plasmacytoid Dendritic Cells Exerts an Antiviral Effect on Thymic HIV-1 Infection
J. Immunol.,
December 15, 2004;
173(12):
7269 - 7276.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Chicha, D. Jarrossay, and M. G. Manz
Clonal Type I Interferon-producing and Dendritic Cell Precursors Are Contained in Both Human Lymphoid and Myeloid Progenitor Populations
J. Exp. Med.,
December 6, 2004;
200(11):
1519 - 1524.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Le Friec, F. Gros, Y. Sebti, V. Guilloux, C. Pangault, R. Fauchet, and L. Amiot
Capacity of myeloid and plasmacytoid dendritic cells especially at mature stage to express and secrete HLA-G molecules
J. Leukoc. Biol.,
December 1, 2004;
76(6):
1125 - 1133.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. ten Hove, F. O. The, M. Berkhout, J. P. Bruggeman, F. A. Vyth-Dreese, J. F. M. Slors, S. J. H. van Deventer, and A. A. te Velde
Expression of CD45RB functionally distinguishes intestinal T lymphocytes in inflammatory bowel disease
J. Leukoc. Biol.,
June 1, 2004;
75(6):
1010 - 1015.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Vanbervliet, N. Bendriss-Vermare, C. Massacrier, B. Homey, O. de Bouteiller, F. Briere, G. Trinchieri, and C. Caux
The Inducible CXCR3 Ligands Control Plasmacytoid Dendritic Cell Responsiveness to the Constitutive Chemokine Stromal Cell-derived Factor 1 (SDF-1)/CXCL12
J. Exp. Med.,
September 2, 2003;
198(5):
823 - 830.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Corcoran, I. Ferrero, D. Vremec, K. Lucas, J. Waithman, M. O'Keeffe, L. Wu, A. Wilson, and K. Shortman
The Lymphoid Past of Mouse Plasmacytoid Cells and Thymic Dendritic Cells
J. Immunol.,
May 15, 2003;
170(10):
4926 - 4932.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Sanz, M. Alvarez-Mon, C. Martinez-A, and A. de la Hera
Human cord blood CD34+Pax-5+ B-cell progenitors: single-cell analyses of their gene expression profiles
Blood,
May 1, 2003;
101(9):
3424 - 3430.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Asnafi, K. Beldjord, E. Boulanger, B. Comba, P. Le Tutour, M.-H. Estienne, F. Davi, J. Landman-Parker, P. Quartier, A. Buzyn, et al.
Analysis of TCR, pTalpha , and RAG-1 in T-acute lymphoblastic leukemias improves understanding of early human T-lymphoid lineage commitment
Blood,
April 1, 2003;
101(7):
2693 - 2703.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Schotte, M.-C. Rissoan, N. Bendriss-Vermare, J.-M. Bridon, T. Duhen, K. Weijer, F. Briere, and H. Spits
The transcription factor Spi-B is expressed in plasmacytoid DC precursors and inhibits T-, B-, and NK-cell development
Blood,
February 1, 2003;
101(3):
1015 - 1023.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. E. Hirst, M. S. Buzza, C. H. Bird, H. S. Warren, P. U. Cameron, M. Zhang, P. G. Ashton-Rickardt, and P. I. Bird
The Intracellular Granzyme B Inhibitor, Proteinase Inhibitor 9, Is Up-Regulated During Accessory Cell Maturation and Effector Cell Degranulation, and Its Overexpression Enhances CTL Potency
J. Immunol.,
January 15, 2003;
170(2):
805 - 815.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M.-C. Rissoan, T. Duhen, J.-M. Bridon, N. Bendriss-Vermare, C. Peronne, B. d. S. Vis, F. Briere, and E. E. M. Bates
Subtractive hybridization reveals the expression of immunoglobulinlike transcript 7, Eph-B1, granzyme B, and 3 novel transcripts in human plasmacytoid dendritic cells
Blood,
October 16, 2002;
100(9):
3295 - 3303.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Ferrero, W. Held, A. Wilson, F. Tacchini-Cottier, F. Radtke, and H. R. MacDonald
Mouse CD11c+ B220+ Gr1+ plasmacytoid dendritic cells develop independently of the T-cell lineage
Blood,
September 26, 2002;
100(8):
2852 - 2857.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. R. Comeau, A.-R. Van der Vuurst de Vries, C. R. Maliszewski, and L. Galibert
CD123bright Plasmacytoid Predendritic Cells: Progenitors Undergoing Cell Fate Conversion?
J. Immunol.,
July 1, 2002;
169(1):
75 - 83.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Weijer, C. H. Uittenbogaart, A. Voordouw, F. Couwenberg, J. Seppen, B. Blom, F. A. Vyth-Dreese, and H. Spits
Intrathymic and extrathymic development of human plasmacytoid dendritic cell precursors in vivo
Blood,
April 15, 2002;
99(8):
2752 - 2759.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. G. de Yebenes, Y. R. Carrasco, A. R. Ramiro, and M. L. Toribio
Identification of a myeloid intrathymic pathway of dendritic cell development marked by expression of the granulocyte macrophage-colony-stimulating factor receptor
Blood,
April 15, 2002;
99(8):
2948 - 2956.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. F. Lipscomb and B. J. Masten
Dendritic Cells: Immune Regulators in Health and Disease
Physiol Rev,
January 1, 2002;
82(1):
97 - 130.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. E. Keir, C. A. Stoddart, V. Linquist-Stepps, M. E. Moreno, and J. M. McCune
IFN-{alpha} Secretion by Type 2 Predendritic Cells Up-Regulates MHC Class I in the HIV-1-Infected Thymus
J. Immunol.,
January 1, 2002;
168(1):
325 - 331.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Dzionek, Y. Sohma, J. Nagafune, M. Cella, M. Colonna, F. Facchetti, G. Gunther, I. Johnston, A. Lanzavecchia, T. Nagasaka, 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.,
December 17, 2001;
194(12):
1823 - 1834.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Mohty, D. Jarrossay, M. Lafage-Pochitaloff, C. Zandotti, F. Briere, X.-N. de Lamballeri, D. Isnardon, D. Sainty, D. Olive, and B. Gaugler
Circulating blood dendritic cells from myeloid leukemia patients display quantitative and cytogenetic abnormalities as well as functional impairment
Blood,
December 15, 2001;
98(13):
3750 - 3756.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Chaperot, N. Bendriss, O. Manches, R. Gressin, M. Maynadie, F. Trimoreau, H. Orfeuvre, B. Corront, J. Feuillard, J.-J. Sotto, et al.
Identification of a leukemic counterpart of the plasmacytoid dendritic cells
Blood,
May 15, 2001;
97(10):
3210 - 3217.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Bauer, V. Redecke, J. W. Ellwart, B. Scherer, J.-P. Kremer, H. Wagner, and G. B. Lipford
Bacterial CpG-DNA Triggers Activation and Maturation of Human CD11c-, CD123+ Dendritic Cells
J. Immunol.,
April 15, 2001;
166(8):
5000 - 5007.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Vandenabeele, H. Hochrein, N. Mavaddat, K. Winkel, and K. Shortman
Human thymus contains 2 distinct dendritic cell populations
Blood,
March 15, 2001;
97(6):
1733 - 1741.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Kadowaki, S. Antonenko, and Y.-J. Liu
Distinct CpG DNA and Polyinosinic-Polycytidylic Acid Double-Stranded RNA, Respectively, Stimulate CD11c- Type 2 Dendritic Cell Precursors and CD11c+ Dendritic Cells to Produce Type I IFN
J. Immunol.,
February 15, 2001;
166(4):
2291 - 2295.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Banchereau, B. Pulendran, R. Steinman, and K. Palucka
Will the Making of Plasmacytoid Dendritic Cells in Vitro Help Unravel Their Mysteries?
J. Exp. Med.,
December 18, 2000;
192(12):
f39 - f44.
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Blom, S. Ho, S. Antonenko, and Y.-J. Liu
Generation of Interferon {alpha}-Producing Predendritic Cell (Pre-Dc)2 from Human Cd34+ Hematopoietic Stem Cells
J. Exp. Med.,
December 18, 2000;
192(12):
1785 - 1796.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Spits, F. Couwenberg, A. Q. Bakker, K. Weijer, and C. H. Uittenbogaart
Id2 and Id3 Inhibit Development of Cd34+ Stem Cells into Predendritic Cell (Pre-Dc)2 but Not into Pre-Dc1: Evidence for a Lymphoid Origin of Pre-Dc2
J. Exp. Med.,
December 18, 2000;
192(12):
1775 - 1784.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Schmitt, H. Fohrer, S. Beaudet, P. Palmer, M.-J. Alpha, B. Canque, J. C. Gluckman, and A. H. Dalloul
Identification of mature and immature human thymic dendritic cells that differentially express HLA-DR and interleukin-3 receptor in vivo
J. Leukoc. Biol.,
December 1, 2000;
68(6):
836 - 844.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
B. Canque, S. Camus, A. Dalloul, E. Kahn, M. Yagello, C. Dezutter-Dambuyant, D. Schmitt, C. Schmitt, and J. C. Gluckman
Characterization of dendritic cell differentiation pathways from cord blood CD34+CD7+CD45RA+ hematopoietic progenitor cells
Blood,
December 1, 2000;
96(12):
3748 - 3756.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Martin, G. M. del Hoyo, F. Anjuere, S. R. Ruiz, C. F. Arias, A. R. Marin, and C. Ardavin
Concept of lymphoid versus myeloid dendritic cell lineages revisited: both CD8alpha - and CD8alpha + dendritic cells are generated from CD4low lymphoid-committed precursors
Blood,
October 1, 2000;
96(7):
2511 - 2519.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y.-J. Liu and B. Blom
Introduction: TH2-inducing DC2 for immunotherapy
Blood,
April 15, 2000;
95(8):
2482 - 2483.
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION