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IMMUNOBIOLOGY
From The Walter and Eliza Hall Institute of Medical
Research, P.O. Royal Melbourne Hospital, 3050 Melbourne, Victoria,
Australia.
In this study, 2 distinct populations of mature dendritic cells
(DCs) were identified in the human thymus. The major population is CD11b Dendritic cells (DCs) are professional
antigen presenting cells1 that form a dynamic network
throughout most tissues and organs and that are crucial for the immune
surveillance of the body.2-4 The current model is that
Langerhans cells, immature DCs found in epithelia, are the precursors
of mature "interdigitating" DCs, found in the T-cell zones of
lymphoid organs.5 Immature interstitial DCs in various
tissues, such as the dermis, move into germinal centers as germinal
center DCs (GCDCs). In the lymph nodes, tonsils, and spleen, the mature
DCs present the antigen captured in the periphery to naive T cells and
induce immunity. The DCs of the thymus have a somewhat different role,
namely, to present self-antigens and induce negative selection of
potential auto-reactive T-cell clones.6 Until recently,
the interdigitating DCs were considered a single population of mature
DCs. However, this laboratory and others showed that distinct DC
subsets of different lineage derivation and different
functions7-13 could be distinguished in mouse lymphoid
organs. On the one hand, CD8+ DCs, lacking myeloid markers
such as CD11b and apparently arising from a lymphoid-committed
progenitor,14 are typically potent interleukin 12 (IL-12)-secreting mature DCs.10,13,15 CD8+
DCs are present at various levels in all mouse lymphoid organs and
constitute the major population in the thymus.15 On the other hand, CD8 Whereas in mice our concepts of DC life history rely mainly on in vivo
studies, investigations of mature human DCs are based mainly on in
vitro models of DC generation. Several human DC precursors have been
identified, including peripheral blood monocytes17-20 and
earlier CD34+ hematopoietic progenitors.21,22
All were shown to differentiate into DCs exhibiting high levels of
major histocompatibility complex class II and costimulatory molecules
upon culture with granulocyte/macrophage-colony stimulating factor
(GM-CSF) and IL-4 or tumor necrosis factor Studies of mature DCs in humans ex vivo31-36 have been
hampered by the difficulty of isolating these cells. Often, short-term culture steps were required to overcome this problem. In the present study, we use freshly isolated DCs to give an instant picture of a
human DC network, adapting a purification procedure used in the
mouse15 to human tissue. We have studied human thymus, which represents a good source of mature DCs in terms of the yield and
the consistency between donors. We have attempted to compare the mature
DC forms found in vivo with those generated in vitro and to compare
human with mouse thymus DCs. We provide an extensive phenotype and
characterization of these DCs and show that 2 distinct populations of
mature DCs, segregated on the basis of CD11b expression, can be
isolated from the human thymus. The major population does not express
CD11b and shares similarities with thymic plasmacytoid-derived DCs
grown in the presence of IL-3, CD40 ligand (CD40L) plus GM-CSF, whereas the minor DC population is CD11b+, arises from
immature myeloid DCs, and is related to tonsillar GCDCs.
Monoclonal antibodies
Immunohistological localization of thymic DCs and
plasmacytoid cells
Four-color immunofluorescence staining and flow cytometry After incubation with directly labeled antibodies, cells were washed twice and stained with streptavidin-Texas Red (Amersham Life Science, Buckinghamshire, United Kingdom) when a biotin-conjugated antibody was used. Analysis was carried out by means of a FacStar Plus (Becton Dickinson), and for each marker, a single-stained control was done to adjust color compensations. At least 8 × 104 events were analyzed per sample.DC purification Human thymus samples were discarded tissue from young children undergoing corrective cardiac surgery and were obtained according to institutional guidelines. The tissue was cut into small fragments, suspended in 10 mL RPMI 1640 containing 2% fetal calf serum (FCS); collagenase (1 mg/mL, type II) (Worthington Biochemical, Freehold, NJ); and DNAse (0.02 mg/mL; grade II bovine pancreatic DNAseI) (Boehringer Mannheim, Mannheim, Germany) and then digested with intermittent agitation for 15 minutes at 37°C followed by 5 minutes at room temperature with constant agitation. To disrupt DC-T-cell complexes, EDTA was added (to 0.01 M final) to the digest, and incubation with agitation was continued for 5 minutes. The suspension was then passed through a stainless steel sieve to remove aggregates and stromal material. All remaining procedures were performed at 0°C to 4°C and used a buffered NaCl/KCl salt solution lacking divalent metals and containing EDTA (EDTA-SS). The cells were recovered from the digest by centrifugation. Then the pellet was immediately resuspended in Nycodenz medium (Nycomed Pharma, Oslo, Norway) (1.068g/cm3 and iso-osmotic with human serum), and a low-density fraction was collected after centrifugation at 1700g for 10 minutes. The low-density fraction was diluted in EDTA-SS, and the cells were recovered by centrifugation. For DC purification, the cells were then incubated for 25 minutes with a mixture of mAbs, including anti-CD3, anti-CD8, anti-CD7, anti-CD15, anti-CD19, anti-CD20, and anti-glycophorinA in EDTA-SS containing 2% human serum. After incubation, the cells coated with mAbs were removed by 2 cycles of sheep anti-mouse immunoglobulin-coupled magnetic beads (Dynabeads, Dynal, Oslo, Norway). The first cycle was at a 3:1 and the second at a 6:1 bead-to-cell ratio. The cells were then kept overnight at 4°C in EDTA-SS containing 10% FCS. The next morning, the cells were incubated for 25 minutes at 4°C with Cy5-conjugated anti-HLA-DR and biotinylated anti-CD11b in EDTA-SS containing 2% human serum. After 2 washes, the cells were incubated with streptavidin-Texas Red. DC populations were then sorted by means of a FacStar Plus. Purity was always superior to 97% after reanalysis. It was determined that this level of staining with anti-HLA-DR antibody did not interfere with T-cell-stimulation assays.Plasmacytoid cell purification For plasmacytoid cell isolation, the undepleted thymic cell preparation was stained with PE-conjugated anti-IL-3R (nonblocking) and Cy5-conjugated anti-HLA-DR. Plasmacytoid cells were sorted on the
basis of high IL-3R and low HLA-DR fluorescence by means of a MoFlow
(Cytomation, Fort Collins, CO). Purity was always superior to 98%
after reanalysis.
Short-term culture of isolated DCs Both thymic DC populations (105) were cultured in 96-well plates (Becton Dickinson Labware, Franklin Lakes, NJ), in 150 µL of complete RPMI (RPMI-1640 supplemented with 10% heat-inactivated FCS; Hepes buffer, pH 7.2; 2 mM glutamine; and 10 4 M 2-mercaptoethanol and antibiotics) containing 7.5 µg/mL of agonistic anti-CD40 (G28.5). After 1 to 3 days of
activation, DCs were either recovered, washed, and incubated with
SS-EDTA for further cell-surface staining, or recovered, washed, and
lysed. Measure of CD11b and CD14 expression after sorting and after
culture was performed by means of a FACScan (Becton Dickinson). A
different clone of anti-CD11b antibody recognizing a different epitope
was used to assess CD11b expression after sorting.
For IL-12 production, isolated DCs (5 × 104 or
105) were cultured in 96 round-bottom plates (Becton
Dickinson Labware) in a final volume of 200 µL complete RPMI
containing recombinant human (rh)GM-CSF (50 ng/mL) (kind gift from Dr
D. Lynch, Immunex, Seattle, WA); rhIL-4 (20 ng/mL) (Peprotech, Rocky
Hill, NJ); rh interferon (IFN)- Culture of isolated plasmacytoid cells Plasmacytoid cells (5 × 104) were cultured in flat-bottom 96-well plates (Becton Dickinson Labware) in 200 µL complete RPMI containing 25 ng/mL rhIL-3 (Peprotech) and 1 µg/mL of soluble trimeric CD40L, with or without 100 ng/mL rhGM-CSF. On day 2 of culture, 100 µL supernatant was recovered for IL-12 enzyme-linked immunosorbent assay (ELISA), and fresh medium was added. On day 5, plasmacytoid-derived DCs were restimulated with either fresh soluble CD40L (1 µg/mL) alone or CD40L (1 µg/mL), rhGM-CSF (100 ng/mL), rhIL-4 (20 ng/mL), and rhIFN- (20 ng/mL). Supernatants were then
recovered on day 7 for IL-12 ELISA.
Reverse transcriptase-polymerase chain reaction Total cytoplasmic RNA was prepared from freshly sorted or activated DCs by means of RNeasy Mini kit (Qiagen, Hilden, Germany). First-strand complementary DNA (cDNA) was synthesized by means of random hexamers, deoxynucleotide triphosphate (dNTP) mixture, rRNAsin ribonuclease inhibitor, and Moloney murine leukemia virus reverse transcriptase (all reagents from Promega, Madison, WI). The polymerase chain reaction (PCR) reaction was carried out in a final volume of 50 µL containing 1 µL cDNA, 1.5 mM MgCl2, thermo-reaction buffer (Promega), dNTP mixture (0.2 mM each dNTP) (Promega), 0.5 mM each oligonucleotide primer (Geneworks Pty, Adelaide, Australia), and 2 U Taq DNA polymerase (Boehringer Mannheim). Each set of 25 to 35 cycles was performed by means of a PerkinElmer thermal cycler (PerkinElmer/Cetus, Norwalk, CT). After 3 minutes of inactivation at 94°C, each cycle consisted of 30 seconds of denaturation at 94°C, 30 seconds of annealing at 60°C, and 30 seconds of extension at 72°C. A final extension at 72°C was carried out for 5 minutes. For analysis, 10 µL of each reaction was electrophoresed through a 2% agarose gel. Monocyte-derived DCs (MoDCs) used as controls were produced from peripheral blood monocytes selected by adherence followed by depletion of contaminants by means of anti-CD3, anti-CD8, anti-CD20, anti-CD15, anti-CD56, and anti-glycophorinA mAbs. Monocytes were then cultured for 6 days with rhGM-CSF (100 ng/mL) and rhIL-4 (20 ng/mL). MoDCs were activated by means of an agonistic anti-CD40 (7.5 µg/mL) for 24 hours. T cells and B cells used as controls were sorted from tonsils on the basis of CD3 and CD20 expression, respectively.The following primers were used for the PCR: T-cell stimulation assays Naive CD4+ T cells were purified from peripheral blood obtained from healthy volunteers. Mononuclear cells were recovered after centrifugation over Ficoll-hypaque (Ficoll-Paque Plus) (Pharmacia Biotech, Uppsala, Sweden). Monocytes/macrophages were subsequently removed by a 1-hour adherence step in RPMI-1640 at 37°C in 150-cm2 culture flasks (Nunc, Roskilde, Denmark) at 106 cells per milliliter. The nonadherent cells were then collected by gently washing the flasks. The naive CD4 T cells were depleted of contaminating cells by incubation with a mixture of mAbs including anti-CD19, anti-CD20, anti-CD45RO, anti-CD8, anti-CD11b, anti-CD15, anti-glycophorinA, and anti-CD56, followed by 2 rounds of Dynabeads (Dynal). The purity of CD4+CD3+CD45RA+ T cells was greater than 90%. Sorted DCs were used as stimulator cells for naive allogeneic CD4 T cells. DCs (10 to 3000) were cocultured with 1.5 × 104 T cells in round-bottom 96-well plates (Becton Dickinson Labware) by means of complete RPMI. After 5 days of coculture, cells were pulsed for 9 hours with 1 µCi 3H-TdR per well and then harvested, and thymidine incorporation was measured by liquid scintillation counting. Assays were performed in triplicate, and results were expressed as mean counts per minute (SD).IL-12 p70 quantitation by ELISA Aliquots of DCs and plasmacytoid cell culture supernatants were assayed by 2 site ELISAs. Briefly, 96-well polyvinyl chloride microtiter plates (Dynatech Laboratories, Chantilly, VA) were coated with purified capture mAb (anti-human IL-12 p70; 20C2) (Pharmingen). Cytokine binding was then detected with a biotinylated detection mAb (anti-human IL-12 p40; C8.6) (Pharmingen). The readout was then obtained by using streptavidin-horseradish peroxidase conjugate (Amersham Life Science) and a substrate solution containing 548 mg/mL ABTS (2,2'-Azino-bis 3-ethylbenz-thiazoline-6-sulfonic acid) (Sigma) and 0.001% hydrogen peroxide (Ajax Chemicals, Auburn, Australia) in 0.1 M citric acid, pH 4.2, followed by scanning the optical density at 405 to 490 nm.
The surface phenotype of mature human thymic DCs In order to study mature thymic DCs, which represent only a small fraction of thymic cells (0.02%), we first enriched the light-density cells (3%) after collagenase digestion and EDTA treatment of the thymic tissue. All detectable DCs were in this fraction. We then carefully depleted non-DC contaminants and in particular CD4CD19+CD20+ B-cell populations,
which expressed surprisingly high levels of costimulatory molecules
(data not shown). Finally, we stained the preparation for
immunofluorescence using 4 colors, analyzed the preparation by flow
cytometry, and focused attention on cells expressing high levels of
HLA-DR as our primary criteria for mature DCs. Other markers were then
used to confirm a mature DC phenotype. As shown in Figure
1, thymic DCs gated on
HLA-DRhighCD86high were also
CD40high and CD83high. Thus, these thymic DCs
displayed a surface phenotype typical of mature DCs. Furthermore,
HLA-DRhighCD86high DCs were all
CD11c+. The broad CD11c profile suggested the existence of
different DC populations but did not allow clear segregation for
further sorting. In contrast, CD11b expression revealed 2 populations of mature DCs: the major population was CD11b, and the
minor one CD11b+. The proportion of the respective
populations varied among donors, but the proportion of the
CD11b DC population was always above 65% of the
HLA-DRhighCD86high mature DCs.
On the basis of this analysis, we adopted the sorting strategy shown in
Figure 2A to collect sufficient numbers
of cells to study both CD11b
As revealed by means of 4-color staining (Figure 2B),
CD11b Both CD11b and CD11b+
DCs, sorted as shown in Figure 2A, proved to be potent stimulators of
naive CD4 T cells. As shown in Figure 4A
and B, CD11b DCs just after sorting displayed a stellate
morphology, typical of fully mature DCs. The CD11b+
fraction (Figure 4D-E) comprised cells of dendritic morphology as well
as "hairy" cells, which were not adherent to glass or plastic. We
observed a rapid clustering (1 to 2 hours) of CD11b DCs
in culture, and after CD40 activation for 24 hours, we observed large
clusters of DCs exhibiting long dendrites (Figure 4C). Freshly sorted
CD11b+ DCs clustered slowly (3 to 5 hours) and, after CD40
activation, formed small clusters of cells displaying long dendrites
(Figure 4F). The morphological features before and after activation,
coupled with the fundamental property of activating naive T cells,
indicated that both CD11b and CD11b+
fractions were DCs, albeit with some differences in form.
CD11b DCs did not
express CD11b or CD14, either when freshly isolated or after 2 days of
CD40 activation by means of an agonistic anti-CD40 antibody. In
parallel, CD11b+ DCs slowly down-regulated CD11b after 2 days of CD40 activation, but they still retained low levels of CD11b
even after 3 days. Importantly, CD14 and CD64 expression was lost early
in culture (less than 24 hours) by the immature CD11b+
fraction, suggesting that these immature DCs needed only CD40 signaling
to mature.
Differences in messenger RNA expression between
CD11b+ and CD11b and
CD11b+ thymic DCs. To ensure we were working with similar
amounts of cDNA from each population, we made dilutions of cDNA samples
to obtain similar expression of the housekeeping gene  -actin. We then compared expression of the maturation marker
DC-LAMP,37 the disintegrin proteinase decysin (which was
identified from CD40-activated GCDCs),40 the TECK
(which was initially described in mouse thymic DCs),41,42
the MIP-1 and the M-CSFR. As shown in Figure 6, DC-LAMP was strongly
expressed by freshly sorted CD11b DCs. In contrast,
freshly sorted CD11b+ DCs expressed little DC-LAMP,
consistent with the fact that this fraction contained a majority of
immature DCs. However, after CD40 activation, CD11b+ DCs
strongly expressed DC-LAMP. Thus, after CD40 activation, both
populations were considered at a similar state of maturation/activation and were not segregated by this marker. Differences between these 2 DC
populations, even after a similar state of activation was attained,
were observed after analysis of decysin and M-CSFR mRNA expression.
Indeed, CD11b+ DCs, but not CD11b DCs,
expressed decysin and the M-CSFR. As expected, decysin expression was
detected only after CD40 activation, when CD11b+ DCs
displayed high expression, whereas only a faint band was observed from
the highest concentration of cDNA from activated CD11b
DCs. The M-CSFR was strongly expressed by freshly isolated
CD11b+ DCs and was still expressed after CD40 activation,
but it was never detected in cDNA from either freshly isolated or
activated CD11b DCs. The chemokine TECK was expressed by
freshly isolated CD11b, but not CD11b+, DCs
although it was down-regulated upon CD40 activation. In contrast,
MIP-1 expression was detected only in cDNA from freshly isolated
CD11b+ DCs, and not in cDNA from CD11b DCs.
The same differences were observed at higher numbers of PCR cycles
(data not shown). These results strongly suggested that
CD11b and CD11b+ DCs represented 2 distinct
thymic DC populations.
Plasmacytoid cells are localized in both the thymic cortex and medulla; mature DCs are localized in the medulla Res et al28 recently showed that in phenotype, thymic plasmacytoid cells resemble their tonsillar and blood counterparts23,24 and that they developed into DCs upon culture with IL-3 and CD40L. Consequently, we tried to determine if one of our thymic DC populations corresponded to the mature dendritic form of plasmacytoid cells. As a first approach, we rechecked the localization of mature DCs and plasmacytoid cells on thymic tissue sections. As shown in Figure 7A, mature DCs expressing high levels of CD40 in blue were found close to Hassall corpuscles, which identify the thymic medulla. Round plasmacytoid cells, identified by high expression of the IL-3R in red, were found
in both the cortex and the medulla, near blue CD40high
DCs displaying typical dendrites (Figure 7B). In contrast to medullary
B cells and epithelial cells, which can express CD40, only DCs
expressed detectable levels of CD86 in blue. As shown in Figure 7C,
blue CD86high DCs were surrounded by round red
IL-3Rhigh plasmacytoid cells in the medulla. Consistent
with our observations by means of flow cytometry, no blue
CD86high DC was found to coexpress the IL-3R.
Thymic plasmacytoid cells can, upon culture, adopt a phenotype
similar to CD11b high and
HLA-DRlow surface expressions (Figure
8A). As shown in Figure 8B,
IL-3Rhigh cells were CD4+,
CD3, CD11c, CD11b,
CD14, CD33, CD20,
CD45RO, CD56, CD16,
CD45RA+, GM-CSFR+, HLA-DR+, and
CD40low and did not express CD86 or CD83. Thus,
IL-3Rhigh thymic cells corresponded in phenotype to
plasmacytoid cells described in tonsils and blood.23,24 We
then sorted thymic plasmacytoid cells using a nonblocking
anti-IL-3R antibody and assessed their further development in
culture with IL-3. As previously described,28 upon culture
with IL-3 and soluble CD40L, plasmacytoid cells clustered after 6 to 8 hours and, after 2 to 3 days, displayed long dendrites (data not
shown). However, it was only after 6 days of culture that
plasmacytoid-derived DCs had matured and acquired CD83 expression
(Figure 9A). Moreover, they then
expressed high levels of HLA-DR and CD86 and still expressed CD45RA,
but did not express CD45RO and CD11c. Thus these IL-3-derived
culture-matured DCs did not correspond to either of our freshly
isolated mature DC populations.
However, in preliminary studies, we found that plasmacytoid cells
derived from thymus and tonsils expressed the GM-CSFR CD11b , IL-4, and soluble
CD40L),43 we stimulated freshly isolated
CD11b and CD11b+ DCs for 1 to 7 days and
measured IL-12 p70 secretion by ELISA. As shown in Table
1, CD11b DCs secreted
substantial amounts of IL-12 p70 after 2 days of stimulation, whereas
CD11b+ DCs were very weak producers regardless of the time
length of culture. Moreover, we tested the ability of thymic
plasmacytoid cells and plasmacytoid-derived DCs to secrete IL-12 p70.
We were not able, in our system, to detect any bioactive IL-12 from the supernatant of plasmacytoid cells cultured for 2 days with IL-3, GM-CSF, and CD40L or from the supernatant of plasmacytoid-derived DCs
restimulated with GM-CSF, IFN-, IL-4, and CD40L on day 5 of
culture.
Our results show that the human thymus contains 2 populations of mature DCs, expressing different lineage markers and
displaying different capacities for IL-12 secretion. The major thymic
DC population (more than 65%) is most readily distinguished from other
DCs by being clearly CD11b In contrast, the minor thymic DC population (less than 35%) is clearly
CD11b+, and also CD11chigh,
CD33high, and CD45ROhigh. Like the major
population, it is CD4+. To collect sufficient numbers of
these cells, we included during sorting not only the mature form of
these cells, but also an immature form expressing intermediate to high
levels of HLA-DR; this immature form was also CD14+,
CD64+, and CD83 The production of IL-12 by DCs has become a major indicator of
functional differences between DC
subpopulations.10,13,25,43,45 In the thymus, negative
selection of self-reactive T cells is performed by DCs6 and
has been shown to be dependent on the presence of IL-12.46
It is therefore of particular interest that we observed a difference
between thymic DC populations in their ability to secrete IL-12. It
should be noted that we found no significative difference by RT-PCR for
TNF- Another important difference is that only the CD11b The origin of these 2 human thymic DC populations and their
relationship to the DC populations in the mouse thymus are issues we
hoped to clarify. Our results provide some pointers, but some contradictions remain. The minor (and variable) CD11b+
thymic DC population appears from its surface markers to be of immediate myeloid origin; such DCs may have entered the thymus directly
from the bloodstream. Although the major CD11b One argument against a plasmacytoid origin for the thymic
CD11b The ability of thymic CD11b
We thank Dr D. Tarlinton and Dr L. Wu for helpful discussions and F. Battye, D. Kaminaris, V. Lapatis, and J. Parker for assistance with flow cytometric sorting. This study is dedicated to the memory of the late Dr Jacques Chiller.
Submitted May 26, 2000; accepted October 23, 2000.
Supported in part by research funding from the University of Melbourne and the Cooperative Research Centre for Vaccine Technology, Queensland Institute of Medical Research, P.O. Royal Brisbane Hospital, Australia (S.V.); and by a Deutsche Krebshilfe fellowship (H.H.).
Preliminary results were presented at the 5th International Symposium on Dendritic Cells, Pittsburgh, PA, September 1998.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Stéphane Vandenabeele, The Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, 3050 Melbourne, Victoria, Australia; e-mail: vandenabeele{at}wehi.edu.au.
1. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol. 1991;9:271-296[CrossRef][Medline] [Order article via Infotrieve]. 2. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245-252[CrossRef][Medline] [Order article via Infotrieve].
3.
Hart DNJ.
Dendritic cells: unique leukocyte populations which control the primary immune response.
Blood.
1997;90:3245-3287 4. Bell D, Young JW, Banchereau J. Dendritic cells. Adv Immunol. 1999;72:255-324[Medline] [Order article via Infotrieve]. 5. Steinman RM, Pack M, Inaba K. Dendritic cells in the T-cell areas of lymphoid organs. Immunol Rev. 1997;156:25-37[CrossRef][Medline] [Order article via Infotrieve].
6.
Brocker T.
Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex class II-expressing dendritic cells.
J Exp Med.
1997;186:1223-1232
7.
Wu L, d'Amico A, Winkel KD, Suter M, Lo D, Shortman K.
RelB is essential for the development of myeloid-related CD8 8. 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. 1997;159:565-573[Abstract].
9.
Reis e Sousa C, Hieny S, Scharton-Kersten T, et al.
In vivo microbial stimulation induces rapid CD40 ligand-independent production of interleukin 12 by dendritic cells and their redistribution to T cell areas.
J Exp Med.
1997;186:1819-1829
10.
Maldonado-Lopez R, De Smedt T, Michel P, et al.
CD8alpha+ and CD8alpha 11. Kronin V, Winkel K, Süss G, et al. A subclass of dendritic cells regulates the response of naive CD8 T cells by limiting their IL-2 production. J Immunol. 1996;157:3819-3827[Abstract].
12.
Süss G, Shortman K.
A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis.
J Exp Med.
1996;183:1789-1796
13.
Pulendran B, Smith JL, Caspary G, et al.
Distinct dendritic cell subsets differentially regulate the class of immune response in vivo.
Proc Natl Acad Sci U S A.
1999;96:1036-1041
14.
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.
1996;184:903-911
15.
Vremec D, Zorbas M, Scollay R, et al.
The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells.
J Exp Med.
1992;176:47-58
16.
Vremec D, Pooley J, Hochrein H, Wu L, Shortman K.
CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen.
J Immunol.
2000;164:2978-2986
17.
Sallusto F, Lanzavecchia A.
Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha.
J Exp Med.
1994;179:1109-1118
18.
Romani N, Gruner S, Brang D, et al.
Proliferating dendritic cell progenitors in human blood.
J Exp Med.
1994;180:83-93
19.
Zhou L-J, Tedder TF.
CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells.
Proc Natl Acad Sci U S A.
1996;93:2588-2592
20.
Randolph GJ, Beaulieu S, Lebecque S, Steinman RM, Muller WA.
Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking.
Science.
1998;282:480-483
21.
Caux C, Vanbervliet B, Massacrier C, et al.
CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNF
22.
Young JW, Szabolcs P, Moore MAS.
Identification of dendritic cell colony-forming units among normal human CD34+ bone marrow progenitors that are expanded by c-kit-ligand and yield pure dendritic cell colonies in the presence of granulocyte/macrophage colony-stimulating factor and tumor necrosis factor alpha.
J Exp Med.
1995;182:1111-1120
23.
Grouard G, Rissoan MC, Filgueira L, Durand I, Banchereau J, Liu YJ.
The enigmatic plasmacytoid T cells develop into dendritic cells with IL-3 and CD40-ligand.
J Exp Med.
1997;185:1101-1111 24. O'Doherty U, Peng M, Gezelter S, et al. Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature. Immunology. 1994;82:487-493[Medline] [Order article via Infotrieve]. 25. Rissoan MC, Soumelis V, Kadowaki N, et al. Reciprocal control of T helper cell and dendritic cell differentiation. Science. 1999;19:1183-1186.
26.
Strobl H, Scheinecker C, Riedl E, et al.
Identification of CD68+lin
27.
Sorg RV, Kogler G, Wernet P.
Identification of cord blood dendritic cells as an immature CD11c
28.
Res PC, Couwenberg F, Vyth-Dreese FA, Spits H.
Expression of pTalpha mRNA in a committed dendritic cell precursor in the human thymus.
Blood.
1999;94:2647-2657
29.
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.
1997;185:875-884
30.
Saunders D, Lucas K, Ismaili J, et al.
Dendritic cell development in culture from thymic precursor cells in the absence of granulocyte/macrophage colony-stimulating factor.
J Exp Med.
1996;184:2185-2196
31.
Hart DNJ, McKenzie JL.
Isolation and characterization of human tonsil dendritic cells.
J Exp Med.
1988;168:157-160 32. Lafontaine M, Landry D, Montplaisir S. The human thymic dendritic cell phenotype and its modification in culture. Cell Immunol. 1992;142:238-251[CrossRef][Medline] [Order article via Infotrieve]. 33. Björck P, Florès-Romo L, Liu YJ. Human interdigitating dendritic cells directly stimulate CD40activated naive B cells. Eur J Immunol. 1997;27:1266-1274[Medline] [Order article via Infotrieve].
34.
Johansson B, Ingvarsson S, Bjorck P, Borrebaeck CA.
Human interdigitating dendritic cells induce isotype switching and IL-13-dependent IgM production in CD40-activated naive B cells.
J Immunol.
2000;164:1847-1854
35.
Takahashi K, Asagoe K, Zaishun J, et al.
Heterogeneity of dendritic cells in human superficial lymph node: in vitro maturation of immature dendritic cells into mature or activated interdigitating reticulum cells.
Am J Pathol.
1998;153:745-755 36. Sotzik F, Rosenberg Y, Boyd AW, et al. Assessment of CD4 expression by early T precursor cells and by dendritic cells in the human thymus. J Immunol. 1994;152:3370-3377[Abstract]. 37. de Saint-Vis B, Vincent J, Vandenabeele S, et al. A novel lysosome-associated membrane glycoprotein, DC-LAMP, induced upon DC maturation, is transiently expressed in MHC class II compartment. Immunity. 1998;9:325-336[CrossRef][Medline] [Order article via Infotrieve]. 38. Sallusto F, Palermo B, Lenig D, et al. Distinct patterns and kinetics of chemokine production regulate dendritic cell function. Eur J Immunol. 1999;29:1617-1625[CrossRef][Medline] [Order article via Infotrieve].
39.
Ito T, Inaba M, Inaba K, et al.
A CD1a+/CD11c+ subset of human blood dendritic cells is a direct precursor of Langerhans cells.
J Immunol.
1999;163:1409-1419
40.
Mueller CGF, Rissoan MC, Salinas B, et al.
PCR selects a novel disintegrin-proteinase from CD40-activated germinal center dendritic cells.
J Exp Med.
1997;186:655-663 41. Vicari AP, Figueroa DJ, Hedrick JA, et al. TECK: a novel CC chemokine specifically expressed by thymic dendritic cells and potentially involved in T cell development. Immunity. 1997;7:291-301[CrossRef][Medline] [Order article via Infotrieve]. 42. Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity. 2000;12:121-127[CrossRef][Medline] [Order article via Infotrieve].
43.
Hochrein H, O'Keeffe M, Luft T, et al.
Interleukin 4 is a major regulatory cytokine governing bioactive interleukin 12 production by mouse and human dendritic cells.
J Exp Med.
2000;192:823-833 44. Grouard G, Durand I, Filgueira L, Banchereau J, Liu YJ. Dendritic cells capable of stimulating T cells in germinal centers. Nature. 1996;384:364-367[CrossRef][Medline] [Order article via Infotrieve]. 45. Cella M, Jarrossay D, Facchetti F, et al. Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nat Med. 1999;5:919-923[CrossRef][Medline] [Order article via Infotrieve].
46.
Ludviksson BR, Ehrhardt RO, Strober W.
Role of IL-12 in intrathymic negative selection.
J Immunol.
1999;163:4349-4359
47.
Wilkinson B, Owen JJ, Jenkinson EJ.
Factors regulating stem cell recruitment to the fetal thymus.
J Immunol.
1999;162:3873-3881
48.
Campbell JJ, Pan J, Butcher EC.
Cutting edge: developmental switches in chemokine responses during T cell maturation.
J Immunol.
1999;163:2353-2357
49.
Res P, Martinez-Caceres E, Jaleco AC, et al.
CD34+ CD38dim cells in the human thymus can differentiate into T, natural killer, and dendritic cells but are distinct from pluripotent stem cells.
Blood.
1996;87:5196-5206
50.
Marquez C, Trigueros C, Franco JM, et al.
Identification of a common developmental pathway for thymic natural killer cells and dendritic cells.
Blood.
1998;91:2760-2771 51. Kelly KA, Lucas K, Hochrein H, Metcalf D, Wu L, Shortman K. Development of dendritic cells in culture from human and murine precursor cells. Cell Mol Biol. In press.
52.
Dalloul AH, Patry C, Salamero J, Canque B, Grassi F, Schmitt C.
Functional and phenotypic analysis of thymic CD34+CD1a 53. Winkel K, Sotzik F, Vremec D, Cameron PU, Shortman K. CD4 and CD8 expression by human and mouse thymic dendritic cells. Immunol Lett. 1994;40:93-99[CrossRef][Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
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  J. Li, J. Park, D. Foss, and I. Goldschneider Thymus-homing peripheral dendritic cells constitute two of the three major subsets of dendritic cells in the steady-state thymus J. Exp. Med., March 16, 2009; 206(3): 607 - 622. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. I. Proietto, S. van Dommelen, P. Zhou, A. Rizzitelli, A. D'Amico, R. J. Steptoe, S. H. Naik, M. H. Lahoud, Y. Liu, P. Zheng, et al. Dendritic cells in the thymus contribute to T-regulatory cell induction PNAS, December 16, 2008; 105(50): 19869 - 19874. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Caminschi, A. I. Proietto, F. Ahmet, S. Kitsoulis, J. Shin Teh, J. C. Y. Lo, A. Rizzitelli, L. Wu, D. Vremec, S. L. H. van Dommelen, et al. The dendritic cell subtype-restricted C-type lectin Clec9A is a target for vaccine enhancement Blood, October 15, 2008; 112(8): 3264 - 3273. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Dasgupta, J. Bayry, S. Andre, J. D. Dimitrov, S. V. Kaveri, and S. Lacroix-Desmazes Auditing Protein Therapeutics Management by Professional APCs: Toward Prevention of Immune Responses against Therapeutic Proteins J. Immunol., August 1, 2008; 181(3): 1609 - 1615. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Varas, C. Hernandez-Lopez, J. Valencia, S. Mattavelli, V. G. Martinez, L. Hidalgo, C. Gutierrez-Frias, A. G. Zapata, R. Sacedon, and A. Vicente Survival and function of human thymic dendritic cells are dependent on autocrine Hedgehog signaling J. Leukoc. Biol., June 1, 2008; 83(6): 1476 - 1483. [Abstract] [Full Text] [PDF]
|
|
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 |
|
 R. K. Reeves and P. N. Fultz Characterization of Plasmacytoid Dendritic Cells in Bone Marrow of Pig-Tailed Macaques Clin. Vaccine Immunol., January 1, 2008; 15(1): 35 - 41. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Lepelletier, S. Smaniotto, R. Hadj-Slimane, D. M. S. Villa-Verde, A. C. Nogueira, M. Dardenne, O. Hermine, and W. Savino Control of human thymocyte migration by Neuropilin-1/Semaphorin-3A-mediated interactions PNAS, March 27, 2007; 104(13): 5545 - 5550. [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]
|
|
|
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M. Lindstedt, K. Lundberg, and C. A. K. Borrebaeck Gene Family Clustering Identifies Functionally Associated Subsets of Human In Vivo Blood and Tonsillar Dendritic Cells J. Immunol., October 15, 2005; 175(8): 4839 - 4846. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Garcia, K. R. Prabakar, J. Diez, Z. A. Cao, G. Allende, M. Zeller, R. Dogra, A. Mendez, E. Rosenkranz, U. Dahl, et al. Dendritic Cells in Human Thymus and Periphery Display a Proinsulin Epitope in a Transcription-Dependent, Capture-Independent Fashion J. Immunol., August 15, 2005; 175(4): 2111 - 2122. [Abstract] [Full Text] [PDF]
|
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 M. Gessi, G. Monego, L. Lauriola, N. Maggiano, and F. O. Ranelletti Parathyroid Hormone-related Peptide (hPTHrP) and Parathyroid Hormone-related Peptide Receptor Type 1 (PTHR1) Expression in Human Thymus J. Histochem. Cytochem., August 1, 2005; 53(8): 955 - 962. [Abstract] [Full Text] [PDF]
|
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S. Patterson, H. Donaghy, P. Amjadi, B. Gazzard, F. Gotch, and P. Kelleher Human BDCA-1-Positive Blood Dendritic Cells Differentiate into Phenotypically Distinct Immature and Mature Populations in the Absence of Exogenous Maturational Stimuli: Differentiation Failure in HIV Infection J. Immunol., June 15, 2005; 174(12): 8200 - 8209. [Abstract] [Full Text] [PDF]
|
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 M. O'Keeffe, T. C. Brodnicki, B. Fancke, D. Vremec, G. Morahan, E. Maraskovsky, R. Steptoe, L. C. Harrison, and K. Shortman Fms-like tyrosine kinase 3 ligand administration overcomes a genetically determined dendritic cell deficiency in NOD mice and protects against diabetes development Int. Immunol., March 1, 2005; 17(3): 307 - 314. [Abstract] [Full Text] [PDF]
|
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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]
|
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L. Li, H.-C. Hsu, C. R. Stockard, P. Yang, J. Zhou, Q. Wu, W. E. Grizzle, and J. D. Mountz IL-12 Inhibits Thymic Involution by Enhancing IL-7- and IL-2-Induced Thymocyte Proliferation J. Immunol., March 1, 2004; 172(5): 2909 - 2916. [Abstract] [Full Text] [PDF]
|
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J. Gotter, B. Brors, M. Hergenhahn, and B. Kyewski Medullary Epithelial Cells of the Human Thymus Express a Highly Diverse Selection of Tissue-specific Genes Colocalized in Chromosomal Clusters J. Exp. Med., January 20, 2004; 199(2): 155 - 166. [Abstract] [Full Text] [PDF]
|
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A. K. Palucka, J. Gatlin, J. P. Blanck, M. W. Melkus, S. Clayton, H. Ueno, E. T. Kraus, P. Cravens, L. Bennett, A. Padgett-Thomas, et al. Human dendritic cell subsets in NOD/SCID mice engrafted with CD34+ hematopoietic progenitors Blood, November 1, 2003; 102(9): 3302 - 3310. [Abstract] [Full Text] [PDF]
|
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E. Donskoy and I. Goldschneider Two Developmentally Distinct Populations of Dendritic Cells Inhabit the Adult Mouse Thymus: Demonstration by Differential Importation of Hematogenous Precursors Under Steady State Conditions J. Immunol., April 1, 2003; 170(7): 3514 - 3521. [Abstract] [Full Text] [PDF]
|
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M. O'Keeffe, H. Hochrein, D. Vremec, B. Scott, P. Hertzog, L. Tatarczuch, and K. Shortman Dendritic cell precursor populations of mouse blood: identification of the murine homologues of human blood plasmacytoid pre-DC2 and CD11c+ DC1 precursors Blood, February 15, 2003; 101(4): 1453 - 1459. [Abstract] [Full Text] [PDF]
|
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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]
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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]
|
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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]
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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]
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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; 99(6): 2084 - 2093. [Abstract] [Full Text] [PDF]
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E. J. Soilleux, L. S. Morris, G. Leslie, J. Chehimi, Q. Luo, E. Levroney, J. Trowsdale, L. J. Montaner, R. W. Doms, D. Weissman, et al. Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro J. Leukoc. Biol., March 1, 2002; 71(3): 445 - 457. [Abstract] [Full Text] [PDF]
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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]
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M.-A. Wurbel, M. Malissen, D. Guy-Grand, E. Meffre, M. C. Nussenzweig, M. Richelme, A. Carrier, and B. Malissen Mice lacking the CCR9 CC-chemokine receptor show a mild impairment of early T- and B-cell development and a reduction in T-cell receptor gamma delta + gut intraepithelial lymphocytes Blood, November 1, 2001; 98(9): 2626 - 2632. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
20°C. After thawing and remoisturizing in phosphate-buffered saline, sections were
double-stained simultaneously with either mouse immunoglobulin (Ig)-G1
anti-CD40 or mouse IgG1 anti-CD86 (BU63), and mouse IgG2a
anti-IL-3R
