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Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 590-598
Definition of Dendritic Cell Subpopulations Present in the
Spleen, Peyer's Patches, Lymph Nodes, and Skin of the Mouse
By
Fabienne Anjuère,
Pilar Martín,
Isabel Ferrero,
Marta López Fraga,
Gloria Martínez del Hoyo,
Natalia Wright, and
Carlos Ardavín
From the Department of Cell Biology, Faculty of Biology, Complutense
University, Spain.
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ABSTRACT |
Dendritic cells (DC) are highly efficient antigen-presenting cells
(APC) that have an essential function in the development of immune
responses against microbial pathogens and tumors. Although during the
past few years our understanding of DC biology has remarkably
increased, a precise characterization of the different DC
subpopulations remains to be achieved with regard to their phenotype
and lineage relationships. In this report, we have extensively studied
the DC subpopulations present in the thymus, spleen, Peyer's patches,
lymph nodes (LN) and skin of the mouse. Thymus DC and 60% spleen DC
have a lymphoid DC phenotype, ie, CD8+
DEC-205high Mac-1low, whereas 40% spleen DC
have a myeloid DC phenotype, ie, CD8
DEC-205low Mac-1high. Both CD8+
and CD8 DC are leukocyte function-associated antigen-1
(LFA-1)high and highly adherent. Within Peyer's patches
the majority of DC correspond to the CD8+
DEC-205high Mac-1low
lymphoid category. In the LN, together with CD8+ and
CD8 DC, an additional nonadherent CD8int
LFA-1int subpopulation with lymphoid DC characteristics is
described. Finally, in the skin both epidermal Langerhans cells (LC)
and dermal DC are CD8DEC-205high Mac-1
high , and do not express LFA-1. Interestingly, LC
migration experiments indicate that LC underwent the upregulation of
CD8 and LFA-1 upon migration to the LN, supporting the hypothesis that
LC belong to the CD8+ lymphoid lineage.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DENDRITIC CELLS (DC) ARE
antigen-presenting cells (APC) with a key function in the immune system
as initiators of T-cell responses against microbial pathogens and
tumors due to their capacity to stimulate naive T cells.1
During the past few years, DC have become a very active area of
research due to the possibility to use DC for antitumoral
immunotherapy.1-4 In this sense, several reports in the
murine system have described tumor regression mediated by the induction
of antitumor CTL responses after transfer of DC pulsed with tumor
antigens.4 These results provided a promising experimental
basis for the development of clinical trials based on the antitumoral
therapeutic potential of DC.3
Because DC are difficult to isolate in large numbers from lymphoid
tissues, the majority of DC-mediated immunotherapy experiments have
been performed using DC differentiated and expanded in vitro, following
a variety of protocols differing in the DC precursor population and the
cytokine combination employed.1 However, it is important to
take into account that different DC subsets exist within lymphoid
organs, and that DC generated in vitro from a defined precursor
population may differ in their phenotype and more importantly in their
APC potential, depending on the cytokines used. In this sense, to fully
exploit the DC potential for immunotherapy, the best source of DC and
experimental conditions to induce an optimal antitumor T-cell immune
response have to be established. Consequently, a precise definition of
the different DC subpopulations found in lymphoid and nonlymphoid
tissues has to be achieved, with regard to both their T-cell
stimulation capacity, and importantly their corresponding precursors.
Experiments performed particularly in the murine system, have allowed
the definition of two main DC subtypes that have been termed lymphoid
and myeloid DC on the basis of their expression of the lymphoid and
myeloid markers CD8 and Mac-1, respectively.5,6 In
addition, functional differences between CD8+ and
CD8 splenic DC concerning their T-cell stimulation
potential, phagocytic activity, interleukin (IL)-12 secretion capacity
and localization within the spleen, have been reported.7-10
With regard to their progenitors, although thymic lymphoid DC have been
shown to derive from lymphoid precursors both in the human and murine
systems,11 the precursors of myeloid DC have not yet been
precisely defined. Interestingly, a recent report analyzing the
phenotype of mice homozygous for an Ikaros null mutation (Ikaros
C/ mice)12 in which TCR  T
cells, CD8+ DC, and myeloid cells are produced, but neither
B cells, nor natural killer (NK) cells, nor CD8 DC,
suggest that CD8 DC may be related to the
B-cell/NK-cell lineage rather than to the myeloid lineage. Besides, the
correlation between skin Langerhans cells (LC), considered to be
immature DC, and mature CD8+ and CD8 DC
remains to be clarified. Therefore, additional studies are required to
define the differential APC potential and the lineage relationships of
the various DC subsets. In this context, to further understand the
correlation between the different DC subtypes constituting the DC
system, we have extensively studied the DC subpopulations present in
the thymus, spleen, Peyer's patches, lymph nodes (LN), and skin of the
mouse, by analyzing their specific characteristics concerning their
phenotype, as well as their adherence and migratory capacity.
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MATERIALS AND METHODS |
Animals.
BALB/c and C57BL/6 mice were purchased from IFFA Credo
(L'Arbresle, France). In all experiments 5- to 7-week-old female mice were used.
Isolation of DC from the thymus, spleen, Peyer's patches, and lymph
nodes.
DC were purified from thymus, spleen, Peyer's patches, and mesenteric
and peripheral (auricular, axillar, and inguinal) LN, following an
isolation protocol modified from our previously described method that
avoids DC culture.13,14 Organs were cut into small fragments and then digested with collagenase A (0.5 mg/mL;
Boehringer-Mannheim, Mannheim, Germany) and Dnase I (40 µg/mL,
Boehringer-Mannheim) in RPMI 1640 medium supplemented with 5% fetal
calf serum (FCS) for 10 minutes at 37°C with continuous agitation.
Digested fragments were filtered through a stainless-steel sieve, and
cell suspensions washed twice in phosphate-buffered saline (PBS)
solution supplemented with 5% FCS and 5 mmol/L EDTA (PBS-EDTA-FCS)
containing 5 µg/mL Dnase I. The cells were then resuspended in cold
isoosmotic Optiprep solution (Nyegaard Diagnostics, Oslo, Norway), pH
7.2, density 1.061 g/cm3, containing 5 mmol/L EDTA to
dissociate DC-thymocyte complexes, and a low-density fraction,
accounting approximately for 1% of the starting cell population,
obtained by centrifugation at 1700g for 10 minutes, and washed
twice in PBS-EDTA-FCS. T-lineage cells, B cells, macrophages and
granulocytes were depleted by treating the recovered low-density cells
for 50 minutes at 4°C with a monoclonal antibody (MoAb) mixture
including anti-CD3 (clone KT3-1.1), anti-CD4 (clone GK1.5),
anti-IL-2R (clone PC61.5), antimacrophage antigen F4/80 (clone
C1.A3-1), and anti-granulocyte antigen Gr1 (clone RB6-8C5). The
unwanted cells were then removed magnetically after incubation for 30 minutes at 4°C with a 1:1 mixture of antimouse Ig and antirat Ig
coated magnetic beads, (Dynabeads, Dynal, Oslo, Norway) at a 7:1
bead-to-cell ratio. Flow cytometry analysis of the DC-enriched
preparations obtained by this isolation method showed that they had a
purity greater than 75% for the thymus, spleen, and Peyer's patch DC
preparations, and greater than 90% for the mesenteric and peripheral
LN DC preparations, as assessed by their CD11c expression (data not
shown). Subsequent phenotypic analysis of the different DC subsets was
performed after gating for CD11c+ cells.
LN DC-adherence assay.
Mesenteric and peripheral (auricular, axillar, and inguinal) LN DC were
purified as described, and incubated in RPMI 1640 medium with 5% FCS
for 90 minutes at 37°C in 35-mm Petri dishes. After this culture
time, the nonadherent cells were obtained by carefully collecting the
floating cells. After washing the culture surface with warm RPMI medium
with 5% FSC to remove the remaining nonadherent cells, adherent cells
were collected by gentle pipetting.
Isolation of DC from the skin.
LC were obtained from epidermal sheets of mouse ears following a
protocol modified from Schuler and Steinman.15 Briefly, ears were split with the aid of forceps into dorsal and ventral halves
and incubated with 0.5% trypsin (Sigma, St Louis, MO) in PBS
containing 5% FCS for 30 minutes at 37°C to allow the separation of the epidermal sheets from dermis. Trypsin treatment under these conditions did not affect the expression by LC of trypsin-sensitive markers, such as LFA-1 or L-selectin, as assessed by trypsin-treatment assays performed on mesenteric LN (data not shown), but DEC-205 was
partially degraded, resulting in a reduced expression of this marker,
as previously described.16 However, after overnight culture
DEC-205 expression by LC was restored (Fig 2). Epidermal cell
suspensions were obtained by filtering the trypsinized epidermal sheets
through a stainless-steel sieve, washed in PBS with 5% FCS and a
LC-enriched low-density fraction, accounting for 5% to 10% of the
starting epidermal cell population, obtained by centrifugation in cold
isoosmotic Optiprep solution as described above. This LC-enriched
low-density cell fraction contained 20% to 30% LC, as assessed by
flow-cytometry analysis of CD11c expression (data not shown).
Subsequent phenotypic analysis of LC preparations was performed after
gating for CD11c+ cells.
The comparative phenotypic analysis of epidermal LC and dermal DC shown
in Fig 6 has been performed after purifying these two DC subpopulations
in parallel following a method modified from the one previously
described by Lenz et al.17 Briefly, whole ears were rinsed
with 70% ethanol and the epidermal and dermal sheets were prepared as
above and cultured for 24 hours in 24-well tissue culture plates in the
presence of 100 ng/mL granulocyte-macrophage colony-stimulating factor
(GM-CSF) (GM-CSF was kindly provided by Immunex Corp,
Seattle, WA). After this culture period most LC and DC together with
keratinocytes were released to the culture medium, and a high
proportion of keratinocytes adhered to the plastic surface. The
nonadherent cell fraction was then collected and the low-density
fraction was obtained as described above. The epidermal low-density
fraction obtained by this method contained 60% to 80% LC, whereas the
dermal low-density fraction contained 30% to 40% DC, as assessed by
flow-cytometry analysis (data not shown). Phenotypic analysis of both
skin DC subsets was performed after gating for CD11c+
cells. This method allows to perform in parallel the isolation of both
epidermal LC and dermal DC, in contrast with the protocol described
above, which is designed specifically for the isolation of epidermal LC
but has the advantage over the latter of not involving a 37°C
incubation step. In this sense, it is important to take into account
that the culture period in the presence of GM-CSF included in the
latter method determined some phenotypic variations, as discussed
below.
LC migration assay.
BALB/c mice received 10 µL of 1% fluorescein isothiocyanate (FITC)
(Sigma) dissolved in 1:1 acetone:dibutylphtalate (ADBP), on the dorsum
of both ears following the protocol described by Cumberbatch et
al.18 After 18 hours, 48 hours, or 5 days, the draining
auricular LN DC were isolated as described.
Flow cytometry.
Phenotypic analysis of DC subpopulations was performed after triple
staining with FITC-conjugated anti-CD11c (clone N418, hamster IgG),
phycoerythrin (PE)-conjugated anti-CD8 (clone CT-CD8a, rat IgG2a;
Caltag, San Francisco, CA) and biotin-conjugated anti-Mac-1 (clone
M1/70, rat IgG2b), anti-DEC-205 (clone NLDC-145, rat IgG2a), anti-LFA-1 (clone FD441.8, rat IgG2b), anti-Fc RII/III (clone 2-4G2), anti-B7-2 (clone GL1, rat IgG2a; Pharmingen, San Diego, CA),
anti-CD40 (clone FGK45, rat IgG), anti-L-selectin (clone MEL-14, rat
IgG2a) or anti-macrophage antigen F4/80 (clone 31-A3-1, rat IgG2b)
followed by streptavidin-tricolor (Caltag). Analysis of the phenotype
of FITC+ cells in LC-migration assays was performed after
double staining with PE-conjugated anti-CD8 and biotin-conjugated
anti-CD11c, anti-Mac-1, anti-DEC-205, or anti-LFA-1 followed by
streptavidin-tricolor. Ig isotype-matched control antibody stainings
for rat IgG2a MoAbs were performed with anti-B220 (clone RA3-6B2, rat
IgG2a) and shown in Fig 2. Equivalent background staining profiles were
obtained with the nonreactive MoAbs anti-CD69 (clone H.1.2F3, hamster
IgG) and anti-TCR-V11 (clone RR8.1, rat IgG2b), used as
isotype-matched control antibodies for hamster IgG and rat IgG2b MoAbs,
respectively (data not shown). All the staining steps were performed at
0° to 4°C in PBS containing 5 mmol/L EDTA and 2% FCS. Analysis
was performed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) at the Flow Cytometry Laboratories of the Faculty of Biology
and the Fundación Jiménez Díaz (Madrid, Spain),
using Lysys II and PC-Lysys softwares (Becton Dickinson).
| |
RESULTS |
DC have been characterized on the basis of their antigen presentation
potential and their phenotype in a variety of lymphoid and nonlymphoid
organs, using sophisticated isolation strategies. Although different DC
subsets display specific functional and phenotypic features, they share
a characteristic phenotypical profile: in the mouse, DC are considered
to be MHC II+, CD11c+, B7.2+,
CD40+, HSA+, CD3,
CD4, B220, Ig,
Gr1.11 In addition, as shown in
Fig 1 the differential expression of other
cell surface molecules, such as CD8, Mac-1, DEC-205, and LFA-1 allows a
precise definition of DC subpopulations in the thymus, spleen, Peyer's
patches, LN, and skin of BALB/c mice. Equivalent DC subpopulations can
be defined in C57BL/6 mice, although minor differences in the
proportion of the different splenic and LN DC subpopulations were
observed (data not shown).
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| Fig 1.
Definition of DC subpopulations from the spleen, Peyer's
patches, LN, and skin of the mouse. Contour plots show the CD8 versus
CD11c, DEC-205, LFA-1, or Mac-1 profile of uncultured DC purified from
the spleen, Peyer's patches, mesenteric and peripheral LN, and skin of
BALB/C mice, gated for CD11c+ cells. CD8+
and CD8 DC subpopulations can be defined (A and B) in
the spleen . In the Peyer's patches most DC are of the
CD8+ subset. In the LN an additional CD8int
DC subset exists (C), which can be further subdivided on the basis of
the CD8 versus LFA-1 expression: in the mesenteric LN, around 50%
CD8int DC are CD8int-high LFA-1low
(C1 cells) whereas the remaining 50% are CD8int-low
LFA-1int (C2 cells), whereas in peripheral LN,
CD8int DC constitute a single population of
CD8int-low LFA-1int cells (C2 cells)
(see text for details). Finally, epidermal LC constitute a single DC
subpopulation with a distinctive phenotype. The CD8 versus DEC-205
contour plot for the skin corresponds to LC after overnight incubation
because, as indicated in Materials and Methods, trypsin treatment used
during LC isolation causes partial DEC-205 degradation, which can be
restored on culture at 37°C (see also Fig 2). These data are
representative of five to eight experiments with similar results.
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Spleen DC.
Spleen DC can be subdivided in two splenic DC subpopulations on the
basis of CD8 expression (A and B, Fig 1). CD8+ and
CD8 subsets represent approximately 60% and 40% of
total splenic DC, respectively. CD8+ splenic DC have the
same phenotype than thymic DC (not shown), ie, they are
CD8+, express the endocytic receptor DEC-205 recognized by
the MoAb NLDC-145, display low levels of the myeloid marker Mac-1, and have high levels of LFA-1, FcR, B7-2, and CD40 on their surface (Fig 2). On the other hand,
CD8 splenic DC are DEC-205low
Mac-1high, display high levels of LFA-1 and FcR, and
express the costimulatory molecules B7-2 and CD40 at lower levels than
CD8+ splenic DC. Our results concerning a differential CD40
expression by splenic CD8+ and CD8 DC
are in agreement with Vremec and Shortman,6 although in this report the level of CD40 expression by both splenic DC subsets was
lower than that described here, probably due to differences in the
reagents used to detect this molecule by flow cytometry.
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| Fig 2.
Phenotype of the major murine DC subpopulations. The
histograms show the phenotype of the principal murine DC subpopulations
defined in Fig 1. CD8+ and CD8 DC from the
Peyer's patches and LN have an almost identical phenotype than
CD8+ and CD8- spleen DC respectively (not
shown). The cells were analyzed after triple staining with
FITC-conjugated anti-CD11c, PE-conjugated anti-CD8, and
biotin-conjugated antibodies against the indicated markers followed by
streptavidin-tricolor. The forward scatter (FSC) of the different DC
subpopulations is compared with that of peripheral T cells (black
profiles in the FSC histograms). Grey profiles in the L-selectin
histograms represent the background staining with a nonreactive control
MoAb (biotin-conjugated anti-B220, clone RA3-6B2) for each DC
subpopulation. Details dealing with the different Ig isotype-matched
control antibodies used are given in Materials and Methods. The dotted
profile in the DEC-205 histogram for skin LC represents the DEC-205
expression after overnight incubation. The vertical dotted lines mark
the lower limit defining the expression at high levels of the
corresponding marker. These data are representative of five to eight
experiments with similar results. MES-LN: mesenteric LN; PER-LN:
peripheral LN.
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As discussed below, on the basis of their phenotypic, developmental,
and functional characteristics, CD8+ and
CD8 DC have been defined as lymphoid and myeloid DC,
respectively.6
Peyer's patch DC.
The majority (around 70%) of DC isolated from mouse Peyer's patches
belong to the CD8+ lymphoid category, and consequently
express DEC-205 and LFA-1 at high levels and low levels of Mac-1 (Fig
1). The rest of Peyer's patch DC includes around 10% of
CD8 myeloid DC and around 20% of DC displaying
intermediate levels of CD8. These CD8int DC are similar to
those found in the peripheral LN (see below).
LN DC.
As illustrated in Fig 1 and 2, mesenteric and peripheral LN DCs can be
subdivided in three DC subpopulations. Apart from a CD8+
DEC-205high Mac-1low lymphoid DC subpopulation
and a CD8- DEC-205low Mac-1high
myeloid DC subpopulation equivalent to those described in the spleen, a
third DC subpopulation displaying high levels of DEC-205, and from low
to intermediate levels of CD8, can be defined (C, Fig 1). The
expression of CD8 and DEC-205 by the CD8int LN DC subset
supports the view that it belongs to the lymphoid category. In support
of this hypothesis, in a recent report Inaba et al19 have
shown that LN DC displaying intermediate levels of CD8 are functionally
equivalent to the CD8+ splenic DC.
CD8+, CD8int, and CD8- LN DC
subpopulations represent approximately 20%, 60%, and 20%
respectively in the mesenteric LN, and 20%, 65%, and 15% in the
peripheral LN. Forward scatter analysis showed that lymphoid DC, ie,
CD8+ DC from the thymus, spleen, Peyer's patches, and LN,
as well as CD8int from LN, are bigger than myeloid DC, ie,
CD8- DC from the spleen and LN (Fig 2).
The comparative analysis of CD8int DC from mesenteric and
peripheral LN (Fig 1 and 2, Table 1) showed
that mesenteric LN CD8int DC comprise two subpopulations
defined on the basis of the CD8 versus LFA-1 expression (C1 and C2 in
Fig 1). Around 50% are CD8int-high LFA-1low
(C1 cells) whereas the remaining 50% are CD8int-low
LFA-1int (C2 cells). Both C1 and C2 cells are
Mac-1low FcRlow. On the other hand, peripheral
LN CD8int DC constitute a single population of
CD8int-low LFA-1int cells (C2 cells),
similar to the C2 population described in the mesenteric LN, although
in contrast to the latter, peripheral LN CD8int DC display
intermediate to high levels of Mac-1 and FcR. With regard to C1 and C2
CD8int DC subpopulations described in the mesenteric LNs,
their functional significance is currently being analyzed.
Interestingly, as shown in Fig 3, the
analysis of the adherence capacity of DC purified from mesenteric or
peripheral LN showed that both the CD8+ and
CD8 DC subsets, which display high LFA-1 levels,
were strongly adherent. On the other hand, CD8int DC from
mesenteric LN (including the previously described C1 and C2
subpopulations), as well as CD8int DC from peripheral LN,
were nonadherent. Interestingly, these CD8int nonadherent
DC displayed low to intermediate levels of LFA-1. Additional adherence
assays have shown that thymic DC, and both CD8+ and
CD8 spleen DC, are adherent cells, whereas skin DC
are nonadherent (data not shown).
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| Fig 3.
Phenotype of adherent and nonadherent DC subsets from
mesenteric and peripheral LN. Mesenteric and peripheral LN DC were
purified as described, incubated for 90 minutes at 37°C, and the
nonadherent and adherent fractions collected and analyzed for the
expression of CD8 and LFA-1. The contour plots show the LFA-1 versus
CD8 profiles of adherent and nonadherent DC after gating for
CD11c+ cells. The dotted lines mark the lower limit
defining the expression at high levels of the corresponding marker.
These data are representative of four experiments with similar results.
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Skin DC.
Epidermal DC, known as epidermal LC, constitute a single population
with a distinctive phenotype (Fig 1). LC are CD11c+,
express neither CD8 nor LFA-1, and display high levels of Mac-1 and
FcR, but intermediate levels of B7-2 and CD40 (Fig 2). In addition
after isolation, a majority of LC express low levels of DEC-205, but
around 30% are DEC-205high. As previously
reported16 and as indicated in Materials and Methods,
DEC-205 was partially degraded by the trypsin treatment employed during
the isolation process. However, after overnight culture at 37°C, LC
underwent an upregulation of DEC-205, and all LC became
DEC-205high, displaying this marker at an expression level
comparable to that found on CD8+ DC from the thymus,
spleen, or LN (Fig 1), according to Inaba et al.16
Importantly, trypsin treatment did not affect either CD8 or LFA-1
expression, as shown by trypsin treatment assays performed in the same
conditions on mesenteric LN and by the fact that some lymphocytes
present in the epidermal LC preparations obtained after trypsin
treatment, displayed CD8 and LFA-1 expression levels comparable to that
of untreated peripheral LN lymphocytes (data not shown).
LC have been shown to represent immature DC that differentiate into
mature DC when they migrate to the T-cell areas of the draining lymph
nodes after an antigenic stimulation.20 Because most LN DC
express intermediate to high levels of CD8 and are positive for
DEC-205, it can be speculated that LC might represent immature DC of
the lymphoid type, which acquire a mature lymphoid DC phenotype upon
stimulation and migration. To test this hypothesis, we analyzed the
phenotype of LC migrating to the peripheral draining LN after exposure
to contact-sensitizing chemicals. For this purpose, BALB/c mice
received 10 µL of 1% FITC in ADBP on the dorsum of both ears,
following the method described by Cumberbatch et al.18 After skin sensitization, the phenotype of the FITC+ LC
that had migrated to the draining auricular LN was analyzed by
purifying the DC from the draining LN. As illustrated in
Fig 4, showing the phenotype of purified
auricular LN DC 5 days after skin sensitization, FITC+
cells were exclusively found within the CD8int DC
subpopulation and represent around 20% of total DC from the draining
LN. Similar results were obtained when the LN were analyzed 18 hours or
48 hours after skin sensitization (data not shown). FITC+
DC had an almost identical phenotype than FITC
CD8int DC present in the auricular LN. No FITC+
cells were found within the auricular T-cell or B-cell populations (data not shown). Consequently, these data suggest that at least some
peripheral LN CD8int DC derive from LC that have migrated
to the draining LN from the epidermis, and that LC migration was
accompanied by the upregulation of CD8 and LFA-1, as illustrated in
Fig 5.
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| Fig 4.
Analysis of LC migrating to the draining LN after skin
sensitization. BALB/c mice received 10µL of 1% FITC in ADBP on the
dorsum of both ears and after 5 days the DC from the draining auricular
LN were purified and analyzed. Contour plots show the CD11c versus CD8
profile of purified auricular LN DC, and the correlation between CD8
expression and FITC staining within these cells. FITC + cells were only found within the CD8 int DC subset and
represented around 20% of total auricular LN DCs. Histograms show the
phenotype of CD8 int FITC + DC (3) compared
with that of CD8 high FITC - DC (1) and CD8
int FITC - DC (2). The vertical dotted lines
mark the lower limit defining the expression at high levels of the
corresponding marker. These data are representative of four experiments
with similar results.
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| Fig 5.
Compared phenotype of skin LC and LC after skin
sensitization-induced migration to the draining LN. Contour plots
represent the CD8 versus LFA-1 profiles of uncultured skin LC and
FITC+ LC isolated from draining auricular LN 5 days after
skin sensitization with FITC. Both CD8 and LFA-1 were upregulated by LC
on FITC-induced migration.
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However, because a population of DC located in the dermis has been
described,17 together with epidermal LC, within the human and mouse skin, in our experiments dermal DC could have also been induced to migrate after skin sensitization and could contribute to the
FITC+ DC population found in the draining LN. In fact, it
could be speculated that FITC+ CD8int DC derive
from dermal DC, and not from epidermal LC as we have proposed above.
Nevertheless, as it has been shown that skin sensitization induces
epidermal LC migration to the LN,18,21-23 if only dermal DC
but not epidermal LC were the precursors of FITC+
CD8int cells found in the LN, we would also expect to find
FITC+ CD8 DC corresponding to migrating
FITC+ LC in the draining LN. However, our results clearly
show that FITC+ DC found in the draining LN constitute a
well-defined homogenous CD8int population. In addition, the
compared phenotypic analysis of epidermal and dermal DC shown in
Fig 6 indicates that in the mouse, both
populations have an almost identical phenotype, according to Lenz et
al,17 suggesting a close lineage relationship between them.
More importantly, our data indicate that both skin DC subsets did not
express either CD8 or LFA-1, and therefore they strongly support the
rationale that CD8 and LFA-1 intermediate levels found on at least a
proportion of FITC+ LN DC in LC migration experiments are
the result of CD8 and LFA-1 upregulation by LC.
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| Fig 6.
Comparative phenotypic analysis of epidermal LC and
dermal DC. The histograms show the expression of the indicated markers
by epidermal LC and dermal DC isolated from epidermal and dermal sheets
respectively, after 24-hour culture as described. Note that  represent the FSC. These data are representative of three experiments
with similar results.
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Note that the phenotype of epidermal LC performed on LC isolated from
epidermal sheets after culture, basically coincides with the analysis
performed on freshly isolated LC presented in Fig 2. However, in
agreement with previous reports15,24,25 because the
isolation method employed to isolate in parallel both skin DC subsets
involves a 24-hour incubation step at 37°C, upon culture LC
underwent the upregulation of CD40, B7-2, and MHC II and the
downregulation of Mac-1 and FcR, but their expression of CD8 and LFA-1
remained unchanged (see Fig 2 and 6; data corresponding to B7-2 and MHC
II are not shown).
| |
DISCUSSION |
Although a wide variety of DC subsets have been described in different
lymphoid and nonlymphoid organs of the mouse,26 two main DC
categories, ie, lymphoid and myeloid DC, can be characterized on the
basis of their origin, phenotypic profile, and physiological properties. Lymphoid DC, such as thymic DC or CD8+ splenic
DC, are defined as CD8+, DEC-205high,
Mac-1low, whereas myeloid DC, such as
CD8 splenic DC are CD8,
DEC-205low, Mac-1high. Evidence of the lymphoid
origin of CD8+ DC derives from thymus reconstitution
experiments showing that mouse thymic DC originate intrathymically from
the CD4low lymphoid precursor population that has no
myeloid potential.13 Later, Wu et al5 reported
that CD8+ but not CD8- splenic DC are generated
after intravenous transfer of CD4low lymphoid precursors.
Additional experiments have shown the differential capacity of
CD8+ and CD8 spleen DC to induce the
stimulation of CD4+ and CD8+ peripheral T
cells, to phagocytose zymosan particles, and to secrete
IL-12.7-10 Importantly, attempts to generate in vitro CD8+ splenic DC from CD8 splenic DC have
been unsuccessful so far, suggesting that the two splenic DC
subpopulations belong, as proposed previously,5 to
different cell lineages and do not represent different differentiation or maturation stages of a unique DC population. In this sense, it has
been recently reported that in mice with a deletion at the C terminus
of the Ikaros gene (Ikaros C /
mice),12 CD8+ but not CD8
splenic DC were generated. Interestingly, these mice displayed some
T-cell differentiation and had a normal development of myeloid cells
but lacked B and NK cells, providing further evidence that CD8
+ DCs are related to the T-cell lineage. Besides, these
data might indicate that CD8 DC do not belong to the
myeloid lineage but rather share some differentiation requirements with
B and NK cells.
The data derived from skin sensitization-mediated LC-migration
experiments showed that some CD8int DC present in the
peripheral LN derive from LC that have migrated from the epidermis, and
that LC migration involves CD8 and LFA-1 upregulation. In this sense,
CD8int DC may represent recent immigrants reaching the LN
as a consequence of an antigenic stimulation and/or a
recirculation process. In this sense, differences in Mac-1 and FcR
expression between mesenteric and peripheral LN CD8int-low
DC may reflect their respective provenance, because mesenteric LN are
known to drain the spleen and intestinal mucosa whereas peripheral LN
drain essentially the skin. With regard to Mac-1 expression, our data
illustrate that upon migration LC partially downregulated this
molecule, although they still expressed Mac-1 at high levels,
comparable to that expressed by CD8int FITC- DC
found in the auricular LN of the FITC-treated mice (Fig 4), or by
CD8int DC from control peripheral LN (Fig 1). Therefore,
the expression of Mac-1 by CD8int DC from the peripheral LN
most likely reflects the fact that at least a proportion of these cells
derive from skin LC that express high levels of Mac-1.
On the other hand, as discussed above, the FITC+
CD8int DC population obtained in the draining LN after skin
sensitization may be result of the migration of both epidermal LC and
dermal DC. The relative contribution of LC to the FITC+
CD8int DC population would most likely be more important
than that of dermal DC because our experiments of skin DC purification,
performed in parallel with epidermal and dermal sheets, indicate that
epidermal LC and dermal DC are present in the adult mouse ear skin at a 5:1 ratio. In support of this rationale, it has been recently reported
that TNF- or oxazolone-induced LC migration to the draining LN was
blocked by antibodies against the 6 integrin subunit, which is
expressed by epidermal LC but not by LN DC.23
Our data strongly suggest that at least a proportion of
FITC+ CD8int LN DC derive from epidermal LC,
and therefore that LC undergo the upregulation of CD8 and LFA-1 upon
migration. Therefore, LC acquire a lymphoid DC-like phenotype upon
migration to the LN, suggesting that they belong to the lymphoid
CD8+ DC lineage. This hypothesis is supported by the fact
that LC have been shown to migrate to the T-cell areas of the lymphoid organs on antigen stimulation,26 and that CD8+
DC have been shown to be located in the T-cell areas of the spleen and
LN.10,19
Concerning LC lineage, classically, LC have been considered to be of
myeloid origin,1 although this hypothesis has not been
formally shown. In fact, DC were also globally considered as myeloid
precursor-derived, until it was shown in the mouse that the
CD8+ DC category derived from lymphoid
precursors.13 In this sense, the origin and precursors of
the so-called myeloid DC, not expressing CD8 in the mouse, also remain
undefined. Similarly, the immediate precursors of LC have not yet been
identified. In the first instance, the observation that in mice
homozygous for a dominant-negative mutation in the Ikaros gene (Ikaros
DN / mice), T cells, B cells and splenic DC
did not develop, whereas LC and myeloid cells were
produced,27 suggested a correlation between LC and the
myeloid lineage. However, the analysis of bone marrow chimeric mice
reconstituted with Ikaros DN / precursor
cells12 showed that Ikaros DN /
mice might have an intrinsic defect in LC/DC differentiation, the
defficiency in the generation of T and B cells being accompanied by a
blockade in DC differentiation at an immature LC stage. Thus the data
derived from Ikaros-deficient mice have not provided an explanation so
far for the correlation between DC and LC lineages. Therefore further
experiments are needed to conclusively determine the origin of
epidermal LC.
In conclusion, our results support the view that lymphoid DC comprise
thymus DC, CD8+ spleen DC, CD8high and
CD8int Peyer's patch and LN DC as well as LC, whereas
CD8 DC from spleen and LN represent the so-called
myeloid DC. Although, as mentioned before, functional differences
between mouse CD8+ and CD8 splenic DC
have been reported,7,8 a precise analysis of the APC
capacity of the different DC subsets has to be achieved, and
importantly, their respective precursors remain to be characterized. In
this sense, although thymus DC have been shown to derive from thymic
CD4low precursors,13 the progenitors of both
CD8+ and CD8 peripheral DC continue to
be largely unknown. The definition of DC precursors might be specially
relevant to define the best source of DC to obtain in vitro the large
number of DC required for antitumor therapeutic purposes. On the other
hand, it is important to take into account that DC differentiated in
vitro may differ in their phenotype and APC function depending on the
culture conditions and on their origin.1 Therefore, the
study of the precursors and function of the different DC subtypes is
crucial to fully exploit the DC tumor immunotherapy potential, because
it can provide the information required to define the most adequate
experimental conditions to induce an optimal DC-mediated T-cell
antitumor response.
| |
ACKNOWLEDGMENT |
The authors thank Anton Rolink for the anti-CD40 hybridoma FGK45 (Basel
Institute for Immunology, Basel, Switzerland), and the Flow Cytometry
facility of the Fundación Jiménez Díaz for making
it possible to perform FACS analysis after hours.
| |
FOOTNOTES |
Submitted March 4, 1998;
accepted October 14, 1998.
Supported by a grant from the DGICYT (PB95-0376), Ministerio de
Educación y Ciencia, Spain (C.A.).
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 correspondence to Dr Carlos Ardavín, Department of Cell
Biology, Faculty of Biology, Complutense University, 28040 Madrid,
Spain; email: ardavin{at}bio.ucm.es.
| |
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X. Zhao, E. Deak, K. Soderberg, M. Linehan, D. Spezzano, J. Zhu, D. M. Knipe, and A. Iwasaki
Vaginal Submucosal Dendritic Cells, but Not Langerhans Cells, Induce Protective Th1 Responses to Herpes Simplex Virus-2
J. Exp. Med.,
January 20, 2003;
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[Abstract]
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G. Schiavoni, F. Mattei, P. Sestili, P. Borghi, M. Venditti, H. C. Morse III, F. Belardelli, and L. Gabriele
ICSBP Is Essential for the Development of Mouse Type I Interferon-producing Cells and for the Generation and Activation of CD8{alpha}+ Dendritic Cells
J. Exp. Med.,
December 2, 2002;
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[Abstract]
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T. W. Spahn, H. Herbst, P. D. Rennert, N. Lugering, C. Maaser, M. Kraft, A. Fontana, H. L. Weiner, W. Domschke, and T. Kucharzik
Induction of Colitis in Mice Deficient of Peyer's Patches and Mesenteric Lymph Nodes Is Associated with Increased Disease Severity and Formation of Colonic Lymphoid Patches
Am. J. Pathol.,
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[Abstract]
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S. Weijzen, M. P. Velders, A. G. Elmishad, P. E. Bacon, J. R. Panella, B. J. Nickoloff, L. Miele, and W. M. Kast
The Notch Ligand Jagged-1 Is Able to Induce Maturation of Monocyte-Derived Human Dendritic Cells
J. Immunol.,
October 15, 2002;
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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;
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2852 - 2857.
[Abstract]
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J. M.M. den Haan and M. J. Bevan
Constitutive versus Activation-dependent Cross-Presentation of Immune Complexes by CD8+ and CD8- Dendritic Cells In Vivo
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September 16, 2002;
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G. Miller, V. G. Pillarisetty, A. B. Shah, S. Lahrs, Z. Xing, and R. P. DeMatteo
Endogenous Granulocyte-Macrophage Colony-Stimulating Factor Overexpression In Vivo Results in the Long-Term Recruitment of a Distinct Dendritic Cell Population with Enhanced Immunostimulatory Function
J. Immunol.,
September 15, 2002;
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[Abstract]
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A. T. Kamath, S. Henri, F. Battye, D. F. Tough, and K. Shortman
Developmental kinetics and lifespan of dendritic cells in mouse lymphoid organs
Blood,
August 13, 2002;
100(5):
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[Abstract]
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P. Martin, G. M. del Hoyo, F. Anjuere, C. F. Arias, H. H. Vargas, A. Fernandez-L, V. Parrillas, and C. Ardavin
Characterization of a new subpopulation of mouse CD8alpha + B220+ dendritic cells endowed with type 1 interferon production capacity and tolerogenic potential
Blood,
June 28, 2002;
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[Abstract]
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G. Moron, P. Rueda, I. Casal, and C. Leclerc
CD8{alpha}2 CD11b+ Dendritic Cells Present Exogenous Virus-like Particles to CD8+ T Cells and Subsequently Express CD8{alpha} and CD205 Molecules
J. Exp. Med.,
May 20, 2002;
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M.-T. Wu and S. T. Hwang
CXCR5-Transduced Bone Marrow-Derived Dendritic Cells Traffic to B Cell Zones of Lymph Nodes and Modify Antigen-Specific Immune Responses
J. Immunol.,
May 15, 2002;
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[Abstract]
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F. Aline, D. Bout, and I. Dimier-Poisson
Dendritic Cells as Effector Cells: Gamma Interferon Activation of Murine Dendritic Cells Triggers Oxygen-Dependent Inhibition of Toxoplasma gondii Replication
Infect. Immun.,
May 1, 2002;
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[Abstract]
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A. D. McLellan, M. Kapp, A. Eggert, C. Linden, U. Bommhardt, E.-B. Brocker, U. Kammerer, and E. Kampgen
Anatomic location and T-cell stimulatory functions of mouse dendritic cell subsets defined by CD4 and CD8 expression
Blood,
March 15, 2002;
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[Abstract]
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B. J. Chen, X. Cui, and N. J. Chao
Addition of a second, different allogeneic graft accelerates white cell and platelet engraftment after T-cell-depleted bone marrow transplantation
Blood,
March 15, 2002;
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[Abstract]
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P. Martin, S. R. Ruiz, G. M. del Hoyo, F. Anjuere, H. H. Vargas, M. Lopez-Bravo, and C. Ardavin
Dramatic increase in lymph node dendritic cell number during infection by the mouse mammary tumor virus occurs by a CD62L-dependent blood-borne DC recruitment
Blood,
February 15, 2002;
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1282 - 1288.
[Abstract]
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K. Kawahata, Y. Misaki, M. Yamauchi, S. Tsunekawa, K. Setoguchi, J.-i. Miyazaki, and K. Yamamoto
Peripheral Tolerance to a Nuclear Autoantigen: Dendritic Cells Expressing a Nuclear Autoantigen Lead to Persistent Anergic State of CD4+ Autoreactive T Cells After Proliferation
J. Immunol.,
February 1, 2002;
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S. Bozza, R. Gaziano, A. Spreca, A. Bacci, C. Montagnoli, P. di Francesco, and L. Romani
Dendritic Cells Transport Conidia and Hyphae of Aspergillus fumigatus from the Airways to the Draining Lymph Nodes and Initiate Disparate Th Responses to the Fungus
J. Immunol.,
February 1, 2002;
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G. M. del Hoyo, P. Martin, C. F. Arias, A. R. Marin, and C. Ardavin
CD8alpha + dendritic cells originate from the CD8alpha - dendritic cell subset by a maturation process involving CD8alpha , DEC-205, and CD24 up-regulation
Blood,
February 1, 2002;
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[Abstract]
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D. J. Campbell and E. C. Butcher
Rapid Acquisition of Tissue-specific Homing Phenotypes by CD4+ T Cells Activated in Cutaneous or Mucosal Lymphoid Tissues
J. Exp. Med.,
January 7, 2002;
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M. F. Lipscomb and B. J. Masten
Dendritic Cells: Immune Regulators in Health and Disease
Physiol Rev,
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[Abstract]
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B. J. Chen, X. Cui, G. D. Sempowski, M. E. Gooding, C. Liu, B. F. Haynes, and N. J. Chao
A comparison of murine T-cell-depleted adult bone marrow and full-term fetal blood cells in hematopoietic engraftment and immune reconstitution
Blood,
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A. Bukreyev, I. M. Belyakov, J. A. Berzofsky, B. R. Murphy, and P. L. Collins
Granulocyte-Macrophage Colony-Stimulating Factor Expressed by Recombinant Respiratory Syncytial Virus Attenuates Viral Replication and Increases the Level of Pulmonary Antigen-Presenting Cells
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C. A. Byersdorfer and D. D. Chaplin
Visualization of Early APC/T Cell Interactions in the Mouse Lung Following Intranasal Challenge
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W. Zhong, A. D. Roberts, and D. L. Woodland
Antibody-Independent Antiviral Function of Memory CD4+ T Cells In Vivo Requires Regulatory Signals from CD8+ Effector T Cells
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August 1, 2001;
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S. Henri, D. Vremec, A. Kamath, J. Waithman, S. Williams, C. Benoist, K. Burnham, S. Saeland, E. Handman, and K. Shortman
The Dendritic Cell Populations of Mouse Lymph Nodes
J. Immunol.,
July 15, 2001;
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M. G. Manz, D. Traver, T. Miyamoto, I. L. Weissman, and K. Akashi
Dendritic cell potentials of early lymphoid and myeloid progenitors
Blood,
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[Abstract]
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H. Hochrein, K. Shortman, D. Vremec, B. Scott, P. Hertzog, and M. O'Keeffe
Differential Production of IL-12, IFN-{{alpha}}, and IFN-{{gamma}} by Mouse Dendritic Cell Subsets
J. Immunol.,
May 1, 2001;
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S. Mori, H. Nakano, K. Aritomi, C.-R. Wang, M. D. Gunn, and T. Kakiuchi
Mice Lacking Expression of the Chemokines Ccl21-Ser and Ccl19 (plt Mice) Demonstrate Delayed but Enhanced T Cell Immune Responses
J. Exp. Med.,
January 15, 2001;
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K. Y. Vermaelen, I. Carro-Muino, B. N. Lambrecht, and R. A. Pauwels
Specific Migratory Dendritic Cells Rapidly Transport Antigen from the Airways to the Thoracic Lymph Nodes
J. Exp. Med.,
January 1, 2001;
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J. M.M. den Haan, S. M. Lehar, and M. J. Bevan
Cd8+ but Not Cd8- Dendritic Cells Cross-Prime Cytotoxic T Cells in Vivo
J. Exp. Med.,
December 18, 2000;
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K. Brasel, T. De Smedt, J. L. Smith, and C. R. Maliszewski
Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures
Blood,
November 1, 2000;
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[Abstract]
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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]
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F. Anjuere, G. M. del Hoyo, P. Martin, and C. Ardavin
Langerhans cells develop from a lymphoid-committed precursor
Blood,
September 1, 2000;
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[Abstract]
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C. F. d'Ostiani, G. Del Sero, A. Bacci, C. Montagnoli, A. Spreca, A. Mencacci, P. Ricciardi-Castagnoli, and L. Romani
Dendritic Cells Discriminate between Yeasts and Hyphae of the Fungus Candida albicans: Implications for Initiation of T Helper Cell Immunity in Vitro and in Vivo
J. Exp. Med.,
May 15, 2000;
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F. Radtke, I. Ferrero, A. Wilson, R. Lees, M. Aguet, and H. R. MacDonald
Notch1 Deficiency Dissociates the Intrathymic Development of Dendritic Cells and T Cells
J. Exp. Med.,
April 3, 2000;
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J. Wang, D. P. Snider, B. R. Hewlett, N. W. Lukacs, J. Gauldie, H. Liang, and Z. Xing
Transgenic expression of granulocyte-macrophage colony-stimulating factor induces the differentiation and activation of a novel dendritic cell population in the lung
Blood,
April 1, 2000;
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[Abstract]
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D. Vremec, J. Pooley, H. Hochrein, L. Wu, and K. Shortman
CD4 and CD8 Expression by Dendritic Cell Subtypes in Mouse Thymus and Spleen
J. Immunol.,
March 15, 2000;
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[Abstract]
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Top
Abstract
Introduction