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HEMATOPOIESIS
From the Departments of Immunobiology and Discovery
Research, Immunex Corporation, Seattle, WA.
Murine dendritic cells (DCs) can be classified into at least 2 subsets, "myeloid-related" (CD11bbright,
CD8 Dendritic cells (DCs) capture and process antigens
for presentation to naive T cells and B cells.1 Although
they are relatively rare, DCs are found in a broad range of lymphoid
and nonlymphoid tissues and are considered to be essential for the
rapid, efficient initiation of an immune response. DCs are considered
to arise from either myeloid-committed or lymphoid-committed
progenitors2; however, the specific stages of development
within these lineages are poorly defined, largely owing to a lack of
understanding of which growth factors regulate this process. More
recent studies have demonstrated that at least 2 cytokines, flt3 ligand
(FL) and granulocyte-macrophage colony-stimulating factor (GM-CSF), induce DC expansion in vivo.3-6 Administration of FL to
mice results in large increases in vivo of 2 populations of DCs, which appear to represent myeloid- and lymphoid-related subsets in normal lymphoid tissues.3 In contrast, GM-CSF administration
favors in vivo expansion of only the myeloid-related
subset.4 Studies in gene knockout (KO) mice support the
notion that these cytokines play different roles in normal DC
development. FL-deficient mice have reduced numbers of lymphoid-tissue
DCs.7 In contrast, mice lacking GM-CSF have normal numbers
of DCs in lymphoid tissues,8 but are functionally
deficient at initiating B-cell and T-cell responses.9,10
The role of cytokines in DC development from hematopoietic
precursors may be more easily examined with in vitro culture systems. Inaba et al11 have shown that murine bone marrow (BM)
cells cultured in GM-CSF for 6 to 8 days generate large numbers of
mature DCs. These GM-CSF-derived DCs can be further activated and
enriched by including interleukin (IL)-4.12,13 DCs
derived in GM-CSF plus IL-4 express cell-surface antigens typically
associated with DCs, including DEC205, major histocompatibility complex
(MHC) class II, CD80, and CD86, and demonstrate potent allo-stimulatory activity.13 On the basis of their expression of myeloid
cell-surface antigens such as CD11b, 33D1, and F4/80, as well as the
lack of CD8 Here we describe a novel method of generating large numbers of DCs in
vitro from BM cells cultured in FL alone. Upon activation with
lipopolysaccharide (LPS) or interferon- Mice
Cell preparations
DC cultures BM cells were cultured in CM containing 200 ng/mL (180 units/mL) human FL (Immunex, Seattle, WA) for 9 days at 1 × 106/mL, in 6-well plates (Costar Corning, Cambridge, MA) unless otherwise noted. Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2 in air. DCs were harvested from the cultures by vigorously pipetting and removing nonadherent cells, then washing each well 2 times with room temperature PBS without Ca++ or Mg++ to remove loosely adherent cells, which were pooled with the nonadherent fraction.DCs were also generated in BM cultures supplemented with GM-CSF plus IL-4 as described.16 Briefly, BM was processed as described above, including lysis of red blood cells. BM cells were cultured at 4 × 105/mL in CM containing 20 ng/mL murine GM-CSF (Immunex) and 20 ng/mL murine IL-4 (Immunex) in 6-well plates. On days 3 and 5 of culture, plates were swirled gently before two thirds of the conditioned medium was removed with the nonadherent cells. Fresh GM-CSF and IL-4-containing medium were added back to the cultures. These cultures were harvested for the nonadherent fraction after vigorous pipetting of DC clusters on day 7, and consisted of DCs and monocytes. Activation of the DCs from FL-supplemented cultures was accomplished by
the addition of 10 ng/mL recombinant murine GM-CSF, 1000 U/mL human
IFN- To distinguish between the presence of soluble and membrane-bound factors produced by the BM cells, 6-well transwell plates (0.4-µm filter; Costar) were used. Various concentrations of feeder cells (whole BM) were added to the upper chamber, and the lower chamber contained 6 × 105 BM cells at 2 × 105/mL (low-cell-density BM). The DCs were harvested from the lower chamber after 9 days of culture. In some experiments, FL was excluded or replaced with 100 ng/mL (160 units/mL) murine SCF (Immunex). The bioactivity of FL and SCF was determined using the WWF7 and MO7E cell lines, respectively. Cultures for cytokine neutralization studies were established as above, and neutralizing antibodies were included at the initiation of culture. Rat anti-murine IL-6 monoclonal antibody (mAb) (MP5-20F3) (Pharmingen, San Diego, CA) and the rat immunoglobulin (Ig)-G1 isotype control (Immunex) were used at 1 µg/mL. Rabbit anti-murine GM-CSF polyclonal antiserum (Immunex) was used at a dilution of 1:400, which was sufficient to neutralize 10 ng/mL of recombinant murine GM-CSF (data not shown). Splenic-derived DCs Adult BALB/c or C57/BL/6 female mice were administered 10 µg/d FL for 10 days in endotoxin-free PBS (Gibco) via intraperitoneal injections.3 At 24 hours after the last injection, the spleens were harvested and the DCs enriched as previously described.17 Briefly, spleens were digested with 100 U/mL collagenase (Worthington Biochemical Corp, Freehold, NJ), in Hanks' buffered salt solution (HBSS, Gibco) with Ca++ and Mg++ and 1% FBS for 30 minutes at 37°C. The spleens were minced and large debris was filtered out before washing the single-cell suspension in HBSS without Ca++ and Mg++ and 10 mmol/L EDTA. For functional assays (mixed lymphocyte reaction [MLR] and stimulation of ovalbumin [OVA]-specific T cells), DCs were enriched by being run over a Nycodenz discontinuous gradient. Cells within the interface were collected and washed twice before being used in antigen-presenting cell (APC) function assays. There were fewer than 1% contaminating T cells or B cells from the DC-enriched interface.Cytologic assays Harvested cells were centrifuged at room temperature onto slides at 30 000 to 40 000 cells per slide. Slides were air-dried and stained with LeukoStat (Fisher Diagnostics, Pittsburgh, PA) for morphological analysis. Phase-contrast observations of cultures were made by means of an inverted microscope (Nikon, Los Angeles, CA) at 400 × magnification.Flow cytometric analysis DCs from harvested cultures were centrifuged once and resuspended in cell-staining medium (SM) consisting of PBS supplemented with 2% heat-inactivated calf serum (Gibco), 2% heat-inactivated mouse serum (Biocell, Rancho Dominguez, CA), 10 µg/mL 2.4G2 anti-Fc receptor mAb (ATCC, Rockville, MD), and 0.02% sodium azide (Sigma, St Louis, MO). Cells were blocked with SM at 4°C for 20 minutes before incubation with mAbs. Cells were incubated with directly conjugated mAbs for 25 minutes at 4°C at 3 to 5 × 105 cells per sample in a 50-µL volume. All mAbs were purchased from Pharmingen, except where noted. The following mAbs (clone name given in parentheses) were used: CD1d (1B1), CD8 (53-6.7), CD11b (M1/70),
CD11c (HL3), CD14 (rmC5-3), CD25 (7D4), CD40 (HM40-3), CD80 (16-10A1),
CD86 (GL-1), IAb (AF6-120.1), Gr-1 (RB6-8C5), Ly6c (AL-21),
Ly6A/E (also known as Sca-1) (D7), and c-kit (ACK45).
Phycoerytherin-conjugated F4/80 (CI:A3-1) mAb was purchased from Caltag
Laboratories (Burlingame, CA). Biotinylated 33D1 (ATCC) and DEC205
(NLDC145; Accurate Chemical and Scientific Corp, Westbury, NY) binding
were detected with streptavidin-phycoerytherin (Molecular Probes,
Eugene, OR). Propidium iodide (Boehringer Mannheim, Indianopolis, IN)
at 2 µg/mL was added to exclude dead cells from analysis. We
collected 10 000 events per sample. Flow cytometric analysis was
performed on a FACSCalibur (Becton Dickinson, Mountain View, CA) with
CELLQuest software (Becton Dickinson). Gates were determined with the
use of appropriate isotype controls. Results are given as the
percentage positive minus background from appropriate isotype controls.
Generation of DCs from sorted progenitor cells BM cells from adult female C57BL/6 mice were processed as above, but resuspended in SM after lysis of red blood cells. Depletion of lineage-positive cells was accomplished by incubating cells with unconjugated mAb to CD11b, CD122 (TMb1), B220 (RA3-6B2), Gr-1, NK1.1 (PK136), TER-119, and F4/80, for 25 minutes at 4°C. All antibodies were from Pharmingen, except F4/80 which was purchased from Caltag Laboratories. Labeled BM cells were washed and then incubated with sheep-antirat IgG-conjugated magnetic beads (Dynal Inc, Oslo, Norway) per instructions. Nonadherent cells were collected and washed, then stained with phycoerythrin-labeled anti-flt3 (A2F10.1) mAb and pooled fluorescein-isothiocyanate-labeled mAb to the following lineage markers (Pharmingen) for 25 minutes at 4°C: CD3 (500A2),
CD11b, CD11c, B220, Gr-1, and Ly6c. The flt3+,
lineage-negative population was sorted on the FACsVantage
(Becton-Dickinson). Collected cells were cultured in a 200 µL vol per
well at high density (approximately 2.5 × 105 cells per
milliliter) in U-well 96-well plates (Costar). Media consisted of CM
supplemented with 200 ng/mL FL and 10% conditioned medium from
cultured spleen cells. Spleen-cell-conditioned medium, a source of
DC-promoting activity,18 was prepared by mincing spleens
from adult C57BL/6 mice in PBS, pipetting them vigorously to break up
clumps, and then washing and resuspending cells in CM. Spleen cells
were then cultured at 3 × 106 cells per milliliter in
T-75 flasks (Costar) for 10 to 14 days before collecting the
conditioned media. After sorted progenitor cells were cultured for 2 days, each well was transferred to another well in a 24-well plate
(Costar) containing FL-and 10%-conditioned media from spleen cells as
above. Cultures were stimulated with 1 µg/mL E
coli-derived LPS on day 8 and harvested for cell counts and flow
cytometric analysis on day 9.
MLR assay DCs were generated as described above. Briefly, BM cells from C57BL/6 mice were cultured for 9 days in media containing FL alone, in cultures containing FL for 9 days to which LPS or IFN- was added
during the final 24 hours of culture, or in cultures containing GM-CSF
plus IL-4 for 7 days. Additionally, DCs were enriched from the spleens
of mice treated with FL for 10 days. T cells were enriched from spleen
and peripheral lymph nodes (LNs) of DBA/2 mice by depletion of non-T
cells with mAbs against B220 and CD11b, followed by removal of
mAb-coated cells with sheep-antirat IgG-conjugated magnetic beads
(Dynal). DBA/2 T cells at 1 × 105 cells per well were
seeded into U-well 96-well plates (Costar) and cultured with varying
numbers of allogeneic C57BL/6-derived DCs in 200 µL per well CM.
Cultures were maintained for 5 days in CM at 37°C in a humidified
atmosphere containing 5% CO2 in air. Alamar blue
(Biosource, Camarillo, CA) at 20 µL per well was added during the
final 24 hours of culture.19 The optical density of the
assay plates was read at a wavelength of 570 to 600 nm on a Molecular
Devices (Sunnyvale, CA) plate reader with the use of SoftMax software
(Molecular Devices).
Preparation and purification of antigen-specific CD4 T cells CD4+ T cells recognizing a peptide of chicken OVA in the context of IAd were isolated from the spleens and peripheral LN of D011.10 TCR transgenic BALB/c mice.20 Single-cell suspensions were incubated for 30 minutes at 4°C with anti-CD8 (53-6.7), MHC class II (IAd, AMS-32.1), and
B220 (RA3-6B2) (Pharmingen). CD4+ T cells were enriched by
depleting mAb-coated cells with sheep-antirat IgG-conjugated magnetic
beads (Dynal).
Antigen-presentation assay OVA presentation assays were performed in 96-well U-well plates (Costar). Naive D011.11 OVA-specific CD4+ T cells at 1 × 105 cells per well were incubated either with a constant number of DCs (2 × 104 cells) per well and titrated OVA protein (Calbiochem, San Diego, CA), or in a constant amount of OVA protein (300 µg/mL) with varying numbers of DCs per well. DCs were generated as described above. Briefly, BM cells from female BALB/c mice were cultured for 9 days in media containing FL alone, or for 9 days in cultures containing FL to which LPS or IFN-
was added during the final 8 hours of culture, or in cultures
containing GM-CSF plus IL-4 for 7 days. Additionally, DCs were enriched
from the spleens of mice treated with FL for 10 days. Cells were
cultured in 200 µL per well in Dulbecco modified Eagle
medium containing 10% FBS, 2-ME, and PSG, at 37°C
in a humidified atmosphere containing 10% CO2 in air. Cells were cultured for 5 days and then pulsed with 0.5 µCi
3H-thymidine for 8 hours, and the cells were then harvested
onto glass fiber sheets for counting in a gas-phase beta counter.
In vitro IL-12 production assay Myeloid- and lymphoid-type DCs from FL-supplemented BALB/c BM cultures were sorted and separated by their expression of CD11b and CD11c by means of the Epics Elite (Beckman Coulter, Brea, CA). Sorted cells were cultured in CM supplemented with 20 ng/mL GM-CSF (Immunex), 20/ng/mL IFN- (PBL, New Brunswick, NJ), and 50 µg/mL
Pansorbin (Calbiochem) at 1 × 106 cells per
milliliter for 40 hours. Culture supernatants were assayed for murine
IL-12 p70 by means of an enzyme-linked immunosorbent assay
(ELISA) Quantikine kit (R & D Systems, Minneapolis, MN). Limit of
detection was 8 pg/mL.
Statistical analysis Statistical analyses were performed by means of the unpaired 2-tailed Student t test.
Generation of CD11c+ DCs from FL-supplemented BM cultures It has previously been reported that the administration of FL to mice generates large numbers of DCs in vivo.3 We were interested in determining whether culture conditions could be established in which FL, as a single factor, could induce DC development in vitro. Murine BM cells were cultured at high density (1 × 106 cells per milliliter) in the presence of 100 ng/mL FL. After 9 days, cultures contained small lymphoid-sized cells that grew in clusters associated with adherent macrophagelike cells. Cells derived from the FL-supplemented cultures of BM cells from BALB/c or C57BL/6 mice expressed variable levels of CD11b, CD11c, 33D1, CD86, MHC class II, and Gr-1 (Figure 1). However, fewer than 1% of the cells were positive for CD19 (B cells), CD3 (T cells), NK1.1 (NK cells), or TER-119 (erythroid
cells) on the basis of flow cytometric analysis (data not shown). The
absence of lineage-specific, cell-surface antigens for T, B, NK, and
erythroid cells, and expression of CD11c and 33D1, as well as low
levels of MHC class II and CD86, were suggestive of immature DC-lineage
committed cells. We detected 2 populations of DCs defined by CD11b
expression. One population expressed high levels of CD11b
(CD11bbright), while the other population expressed little
to no CD11b (CD11bdull). Most of the cells from
FL-supplemented BM cultures were positive for CD11c (76%), with
approximately 50% expressing CD86 or MHC class II. Expression of 33D1,
a marker of marginal zone DCs,21 was restricted to the
CD11bbright population. Gr-1, a marker of granulocytes, was
expressed on half of the CD11b+ FL-derived cells from
BALB/c cultures, but on only a low percentage (7%) of cells from
C57BL/6 cultures. In mice administered FL for 9 days at 10 µg per
mouse per day, we found that splenic-derived CD11c+ DC from
C57BL/6 and BALB/c mice were, respectively, 14% and 25% positive for
Gr-1 (data not shown). Thus, cells generated from FL-supplemented BM
cultures from both C57BL/6 and BALB/c mice generate populations of
cells that are phenotypically similar to DC subsets previously
identified in lymphoid tissues of normal and FL-treated
mice.3,15
FL, but not SCF, supports in vitro DC development Unfractionated BM cells generated DCs in cultures only when the seeding density exceeded 5 × 105/mL (data not shown). Therefore, we investigated the role of endogenously produced soluble factor(s) in the generation of DCs by using a transwell system that allows for the selective diffusion of soluble factors. BM cells at low cell density (2 × 105 cells per milliliter in lower chamber) were cultured with increasing numbers of BM cells (upper chamber) in the presence or absence of FL or SCF, the ligand for c-kit that shares activities and structural homology to FL.22 Regardless of the BM feeder-cell density in the upper chamber, cells cultured at low cell density in the lower chamber did not survive in the absence of exogenous cytokines (Figure 2). Inclusion of SCF in the cultures resulted in a 2-fold expansion of cells in the lower chamber, which was further increased when feeder cells were included in the upper chamber. However, fewer than 4% of the cells from the lower chamber, generated in the SCF-supplemented cultures, expressed CD11c or MHC class II, whereas 87% of the cells expressed high levels of Gr-1 and exhibited the morphology of immature granulocytes (data not shown). BM cells cultured at low density in FL-containing medium generated few cells when the BM feeder cells were excluded (25% of the input number). However, there was a linear dose response in the generation of cells in the lower chamber when increasing numbers of BM feeder cells were included in the top chamber, demonstrating the importance of cell density for the production of soluble factors (Figure 2). Cells harvested from the lower chambers under these conditions expressed CD11c (85%) and MHC class II (70%) similarly to what was shown in Figure 1. Thus, feeder cell-derived soluble factor(s) can support DC development in vitro when FL is the sole growth factor added exogenously.
Role of endogenous IL-6 in DC development from FL-supplemented BM cultures We investigated the role of endogenous soluble factors in DC development in FL-supplemented cultures using neutralizing mAbs. Neutralizing mAbs against IL-2, IL-3, IL-4, IL-7, IL-11, IL-15, granulocyte colony-stimulating factor (G-CSF), CSF-1, and transforming growth factor (TGF)- 1-3 failed to inhibit DC
development in vitro from FL-supplemented BM cultures (data not shown).
Neutralizing mAbs against murine IL-6 inhibited approximately 70% to
80% of the DC development during 9 days of culture (Figure
3A). A similar effect was seen with
neutralizing mAbs against the murine IL-6 receptor (IL-6R) and gp130,
both components of the IL-6 receptor complex (data not shown). In
addition, FL-supplemented cultures of BM cells from IL-6 gene KO mice
produced fewer cells, compared with normal BM, and as expected were not
affected by the inclusion of the neutralizing anti-IL-6 mAb (Figure
3B). Residual cells harvested from FL-supplemented cultures of BM from
IL-6 gene KO mice and control cultures where anti-IL-6 mAb was
included still expressed CD11b, CD11c, CD86, and MHC class II similarly
to control cultures (data not shown). BM cells cultured in recombinant
murine IL-6 alone did not generate DCs (data not shown). Finally,
polyclonal antibody against murine GM-CSF did not affect DC development
in this culture system (Figure 3A). Thus, IL-6, but not GM-CSF, is an
important growth factor for DC development from FL-supplemented BM cultures.
Kinetics of DC development in FL-supplemented BM cultures Cells from FL-supplemented BM cultures were harvested periodically over an 11-day period, counted, and analyzed for cell-surface phenotype. Although the total cellularity was fairly constant over time (1 to 3 × 106 cells per well), there was a considerable change in the relative proportion of monocytic-myeloid and B-lymphoid cells, as assessed by flow cytometry (Figure 4). The number of cells expressing CD19 (B cells) and Gr-1 (primarily granulocytes) declined over time, while there was a concomitant increase in cells expressing CD11c and MHC class II. After 9 days of culture, there were no detectable erythroid cells (TER-119+), T cells (CD3+), or NK
cells (NK1.1+) present (data not shown). Significant
numbers of CD11c+ cells were not present until 5 to 7 days
of culture, and CD11c expression closely correlated with development of
MHC class II expression.
Activation of FL-derived DCs Since the DCs generated from FL-supplemented cultures expressed low levels of DC-associated molecules (Figure 1), we concluded that DCs with an immature phenotype were being generated. We investigated whether these cells could be activated in vitro with cytokines or LPS. DCs generated from FL-supplemented BM cultures were treated with 1 µg/mL E coli-derived LPS, 1000 U/mL IFN-, or 10 ng/mL GM-CSF to assess morphological and phenotypic changes associated with
activation. LPS, IFN-, or GM-CSF was added to FL-supplemented DC
cultures during the final 24 hours of culture (day 8). There was no
statistically significant change in cellularity in the cultures after
24 hours of stimulation with LPS, IFN-, or GM-CSF (data not shown).
Cultures supplemented with FL alone contained small, lymphoid-sized
cells, growing as single-cell suspensions, although some cells grew in
small clusters (fewer than 20 cells per cluster). Some of the cells
also possessed short dendrites (Figure
5A). Cells from LPS-treated cultures were
larger; formed clusters (more than 50 cells per cluster) that adhered
to the plastic culture dishes; and when separated by gentle pipetting, exhibited long dendrites (Figure 5B). Cells from IFN--treated cultures also increased in size and had extended dendrites, but did not
form large adherent clusters (Figure 5C). FL-derived DCs treated with
GM-CSF for 24 hours formed large clusters and increased in size, but
few cells possessed long dendrites (Figure 5D). In addition, for
comparative purposes, we generated DCs in cultures supplemented with
GM-CSF and IL-4 as described.16 BM cells cultured for 7 days in medium supplemented with 20 ng/mL GM-CSF and 20 ng/mL IL-4
consisted primarily of adherent giant-multinucleated cells and
macrophagelike cells, as well as nonadherent cells consisting of monocytes and cells with dendriform bodies. Only those
cells in the nonadherent fraction were used to compare with FL-derived DCs (Figure 5E).
The cell-surface phenotype of activated DCs from FL-supplemented
cultures was assessed by flow cytometry. There was little change in
expression of CD11b or CD11c after 24-hour treatment of cultures with
LPS, IFN-
For comparative purposes, we generated DCs using BM cultures
supplemented with GM-CSF plus IL-4 as previously
described.16 Flow cytometric analysis showed that most of
the cells constitutively expressed high levels of CD11b, CD11c, CD80,
CD86, and MHC class II (Figure 6C). Some of the cells from these
cultures also expressed CD1d, 33D1, CD25, CD40, DEC205, and Sca-1, but
not c-kit or CD8 DCs can be generated from flt3+, lineage-depleted BM cell progenitors To address the question of whether DCs generated from FL-supplemented BM cultures arise from pre-existing DCs or progenitor cells, the following experiment was performed. Cells from the BM expressing flt3, a receptor found on lymphohematopoietic progenitor cells,22 and lacking a number of lineage-specific markers (CD3, CD11b, CD11c, CD122, B220, Gr-1, NK1.1, TER119, F4-80, and
Ly6c) were purified. This flt3+, lineage-depleted
population represented fewer than 0.3% of whole BM. Sorted cells were
cultured in FL plus conditioned medium from cultured spleen cells,
which has previously been described to contain an IL-6-like activity
and could promote murine DC development in vitro.18 After
8 days of culture, the cells were stimulated with LPS and harvested 24 hours later. Addition of LPS was used to up-regulate DC-associated
markers as shown in Figures 6A and 6B. Flow cytometric analysis
demonstrated 2 discrete populations as determined by levels of CD11b
and CD11c (Figure 7). Similarly to DCs
generated from whole BM cultured in FL and stimulated with LPS, most
cells expressed high levels of MHC class II, CD80, CD86, and CD40
(Figures 6A and 7). CD8 and 33D1 were expressed primarily by the
CD11bdull and CD11bbright populations,
respectively (data not shown). Thus, both myeloid- and lymphoid-type
DCs, as assessed by phenotype, could be generated from hematopoietic
progenitors by means of FL and the conditioned medium from
spleen cells.
FL-derived DCs stimulate allogeneic T-cell proliferation DCs from BM cultures supplemented with FL or with GM-CSF plus IL-4, as well as DCs derived from the spleens of mice treated with FL for 10 days, were assayed for allo-stimulatory activity in an MLR assay. Each population of DCs was approximately 75% to 80% positive for CD11c and contained fewer than 1% contaminating T cells (data not shown). All populations of DCs had potent allo-stimulatory activity, although those generated in vitro with FL alone had the least (Figure 8). BM cells cultured in FL and stimulated for 24 hours with IFN- or LPS had enhanced
allo-stimulatory capacity over those in FL alone (approximately
10-fold, at 1000 DCs per well). DCs generated in BM cultures
supplemented with GM-CSF plus IL-4 were comparable in activity to DCs
derived from cultures supplemented with FL and activated with LPS.
Splenic-derived DCs consistently exhibited less activity than in
vitro-derived DCs. Thus, FL-derived DCs activated with IFN- or LPS,
which induced up-regulation of MHC class II and costimulatory
molecules, resulted in potent allo-stimulatory activity to induce
T-cell proliferation in an MLR assay.
Processing and presentation of OVA protein to OVA-specific CD4 T cells We tested the capacity of both FL-derived and GM-CSF plus IL-4-derived DCs to process OVA protein and present it to OVA-specific CD4+ T cells in vitro. T cells from D011.10 transgenic mice express a TCR specific for the OVA peptide fragment 323-339 presented on I-Ad MHC class II molecules.20
OVA-specific CD4+ T cells were enriched from spleens and
LNs of D011.10 mice and then cultured either with varying numbers of
DCs in a constant concentration of OVA protein or with constant numbers
of DCs with titrated OVA protein; proliferation was measured after 5 days. DCs were derived from in vitro cultures containing FL (± LPS) or
GM-CSF plus IL-4, as well as cultures enriched from the spleens of mice
treated with FL for 10 days. All populations of DCs were approximately
72% to 76% positive for CD11c and contained fewer than 1%
contaminating T cells (data not shown). In terms of inducing T-cell
proliferation, DCs derived in vitro with FL and stimulated with LPS
during the final 8 hours of culture demonstrated activity similar to
DCs from cultures supplemented with FL alone; this similarity occurred
both when DCs were titrated and OVA protein remained constant (300 µg/mL) and when OVA protein was titrated and APCs remained
constant (Figure 9A-B).
FL-derived DCs stimulated with IFN- for 8 hours were also used and
found to have activity similar to those stimulated with LPS (data not
shown). When OVA protein was held constant, and APCs were titrated, DCs
generated in vitro with the use of GM-CSF plus IL-4 demonstrated
activity similar to FL-derived DCs. Additionally, those DCs generated
in GM-CSF plus IL-4 were less stimulatory to T cells than FL-derived DCs when DCs were held constant and the OVA protein was titrated (Figure 9B). Splenic-derived DCs enriched from the spleens of mice
treated with FL for 10 days were also used for comparison, but found to
be consistently less stimulatory to T-cell proliferation when compared
with the in vitro-derived DCs. These data demonstrate that DCs
generated in vitro with FL or FL plus IFN- are competent at
processing and presenting OVA protein to OVA peptide-specific CD4+ T cells, and were comparable to those DCs generated in
cultures supplemented with GM-CSF and IL-4.
IL-12 production is restricted to the lymphoid-type DCs Among the most profound functional differences between myeloid- and lymphoid-type DCs is IL-12 production, which is limited to the lymphoid subset.26-28 We asked whether sorted DCs generated in vitro from FL-supplemented cultures could produce IL-12 p70 and, if so, whether it would be restricted to the CD11bdull lymphoid DC subset. DC subsets sorted with the use of the gates as shown in Figure 6B were cultured for 40 hours in the presence of SAC (Pansorbin), GM-CSF, and IFN-.
Lymphoid-type DCs generated from FL-supplemented BM cultures made
10-fold more IL-12 p70 than their myeloid DC counterparts (Table
2). No detectable IL-12 p70 was produced
from unstimulated DCs (data not shown).
Here we describe a novel culture system to generate murine DCs
with cell-surface antigen expression and functional phenotype similar
to those of DCs residing in lymphoid tissues. This culture system
requires the addition of a single growth factor, FL, whereas a
structurally and functionally related growth factor, SCF, promotes the
generation of immature granulocytes. Maximal DC numbers are achieved
after 9 to 10 days of culture, which is similar to the kinetics of
FL-induced DC expansion in vivo.3 The cell-density dependence of this culture system suggests the involvement of endogenous factor(s) as well. Rasko et al29 have
demonstrated the cell-density dependence of murine BM cells in forming
colonies in response to FL alone, which suggests the presence of
endogenous factors. Addition of neutralizing mAb to IL-2, IL-3, IL-4,
IL-7, IL-11, IL-15, CSF-1, G-CSF, GM-CSF, or TGF- GM-CSF has the capacity to induce both DC development from precursor
cells and activation of DCs in vitro and in
vivo.5,6,11,32-43 In the present study, GM-CSF was not
required for DC generation from mouse BM in vitro, which is similar to
a previous report that anti-GM-CSF mAb does not inhibit DC generation
from murine thymic precursors cultured in a cytokine cocktail (TNF- We have demonstrated that as little as 24 hours' exposure to GM-CSF
caused these immature DCs to cluster and up-regulate expression of CD40
and CD80, but there was surprisingly little effect on MHC class II,
CD86, and other DC-associated antigens. Optimal stimulation of
FL-derived DCs with GM-CSF may require longer incubation periods or the
addition of other proinflammatory cytokines. LPS and IFN- CD8 LPS and IFN- The coexpression of both c-kit and Sca-1 following
activation of DCs is a novel observation. Both of these cell-surface
antigens have been previously described as being expressed on lymphoid tissue DC,15,32,43-47 but never together in the same
report. Murine hematopoietic stem cells from C57BL/6 mice are described as lineage marker negative (such as B220, Gr-1, CD11b, CD3 We have also shown that, similarly to FL-treated mice, both lymphoid-
and myeloid-type DCs, as defined by phenotype, are generated when BM is
cultured in vitro with FL.3,26 Myeloid-type DCs are
characterized as CD11bbright, CD11c+,
33D1+, and CD8 One of the pressing issues in murine DC development is whether myeloid-
and lymphoid-type DCs represent separate lineages. Numerous studies
have addressed this issue by culturing progenitors in combinations of
cytokines excluding GM-CSF, or by injecting defined progenitors
directly into mice.14,43,45 However, more recent reports
have indicated that thymic- and splenic-derived lymphoid-type DC
development is not associated with T-cell development since Notch 1 KOs
or mice deficient in both c-kit and IL-2R In an MLR assay, DCs generated in vitro from BM supplemented with FL
and stimulated with LPS had similar allo-stimulatory activity as those
DCs generated in GM-CSF plus IL-4. In addition, DCs generated in FL
plus IFN- Finally, to address whether lymphoid-type DCs generated from
FL-supplemented BM cultures represent those found in lymphoid tissues,
we stimulated both DC subsets to produce IL-12, which has been
described as being restricted to the lymphoid-type subset as defined by
CD11b expression26 or CD8 The culture system described in this report will be useful for our understanding of the development of DCs for their potential use as adjuvants both for infectious disease and in tumor biology.
We thank Gary Carlton, Daniel Hirschstein, and Steve Braddy for technical assistance and Anne Aumell and Drs Hilary McKenna, Stewart Lyman, and Douglas Williams for critical advice and review of this manuscript.
Submitted August 17, 1999; accepted July 5, 2000.
The authors are employees of Immunex Corporation.
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: Kenneth Brasel, Immunobiology Department, Immunex Corporation, 51 University St, Seattle, WA 98101; e-mail: brasel{at}immunex.com.
1. Steinman RM. The dendritic cell system and its role in immunogenity. Annu Rev Immunol. 1991;9:271-296[Medline] [Order article via Infotrieve]. 2. Thomas R, Lipsky PE. Dendritic cells: origin and differentiation. Stem Cells. 1996;14:196-206[Abstract].
3.
Maraskovsky E, Brasel K, Teepe M, et al.
Dramatic increase in the numbers of functionally mature dendritic cells in the flt3 ligand-treated mice: multiple dendritic cell populations identified.
J Exp Med.
1996;184:1953-1962 4. Brasel K, Maraskovsky E, Pulendran B, et al. Preferential expansion of myeloid-type dendritic cells in vivo after administration of GM-CSF into mice: a comparative analysis with Flt3 ligand generated dendritic cells [abstract]. Blood. 1997;90:170a. 5. Hanada K, Tsunoda R, Hamada H. GM-CSFinduced in vivo expansion of splenic dendritic cells and their strong costimulation activity. J Leukoc Biol. 1996;60:181-190[Abstract].
6.
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
7.
McKenna HJ, Stocking KL, Miller RE, et al.
Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells.
Blood.
2000;95:3489-3497 8. Vremec D, Lieschke GJ, Dunn AR, Robb L, Metcalf D, Shortman K. The influence of granulocyte/macrophage colony-stimulating factor on dendritic cell levels in mouse lymphoid organs. Eur J Immunol. 1997;27:40-44[Medline] [Order article via Infotrieve].
9.
Wada H, Noguchi Y, Marino MW, Dunn AR, Old LJ.
T cell functions in granulocyte/macrophage colony-stimulating factor deficient mice.
Proc Natl Acad Sci U S A.
1997;94:12557-12561
10.
Noguchi Y, Wada H, Marino MW, Old LJ.
Regulation of IFN-
11.
Inaba K, Inaba M, Romani N, et al.
Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.
J Exp Med.
1992;176:1693-1702
12.
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
13.
Labeur MS, Roters B, Pers B, et al.
Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage.
J Immunol.
1999;162:168-175
14.
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 15. Vremec D, Shortman K. Dendritic cell subtypes in mouse lymphoid organs. J Immunol. 1997;159:565-573[Abstract].
16.
Garrigan K, Moroni-Rawson P, McMurray C, et al.
Functional comparison of spleen dendritic cells and dendritic cells cultured in vitro from bone marrow precursors.
Blood.
1996;88:3508-3512 17. Crowley M, Inaba K, Witmer-Pack M, Steinman RM. The cell surface of mouse dendritic cells: FACS analyses of dendritic cells from different tissues including thymus. Cell Immunol. 1989;118:108-125[Medline] [Order article via Infotrieve]. 18. Ni K, O'Neill HC. Long-term stromal cultures produce dendritic-like cells. Br J Haematol. 1997;97:710-725[Medline] [Order article via Infotrieve]. 19. Ahmed SA, Gogal RM, Walsh JE. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to H3-thymidine incorporation assay. J Immunol Methods. 1994;170:211-224[Medline] [Order article via Infotrieve].
20.
Murphy KM, Heimberger AB, Loh DY.
Induction by antigen of intrathymic apoptosis of CD4+ and CD8+ TCRlo thymocytes in vivo.
Science.
1990;250:1720-1723 21. Witmer MD, Steinman RM. The anatomy of peripheral lymphoid organs with emphasis on accessory cells: light microscopic immunocyto-chemical studies of mouse spleens, lymph nodes and Peyer's patch. Am J Anat. 1984;170:465-481[Medline] [Order article via Infotrieve].
22.
Lyman SD, Jacobsen SEW.
c-kit ligand and Flt3 ligand: stem/progenitor cell factors with overlapping yet distinct activities.
Blood.
1998;91:1101-1134
23.
Okada S, Nakauchi H, Nagayoshi K, Nishikawa S-I, Miura Y, Suda T.
In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells.
Blood.
1992;80:3044-3050
24.
Li CL, Johnson GR.
Murine hematopoietic stem and progenitor cells, I: enrichment and biological characterization.
Blood.
1995;85:1472-1479
25.
Osawa M, Nakamura K, Nishi N, et al.
In vivo self-renewal of c-kit+ Sca-1+ linlow/
26.
Pulendran B, Lingappa J, Kennedy MK, et al.
Developmental pathways of dendritic cells in vivo.
J Immunol.
1997;159:2222-2231
27.
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
28.
Maldonado-Lopez R, De Smedt T, Michel P, et al.
CD8 29. Rasko JEJ, Metcalf D, Rossner MT, Begley CG, Nicola NA. The flt3/flk-2 ligand: receptor distribution and action on murine haemopoietic cell survival and proliferation. Leukemia. 1995;9:2058-2066[Medline] [Order article via Infotrieve]. 30. Santiago-Schwarz F, Tucci J, Carsons SE. Endogenously produced interleukin 6 is an accessory cytokine for dendritic cell hematopoiesis. Stem Cells. 1996;14:225-231[Abstract].
31.
Schneider E, Ploemacher RE, Navarro S, Beurden C, Dy M.
Characterization of murine hematopoietic progenitor subsets involved in interleukin-3-induced interleukin-6 production.
Blood.
1991;78:329-338
32.
Witmer-Pack MD, Oliver W, Valinsky J, Schuler G, Steinman RM.
Granulocyte/macrophage colony-stimulating factor is essential for the viability and function of cultured murine epidermal Langerhan cells.
J Exp Med.
1987;166:1484-1498
33.
Inaba K, Steinman RM, Witmer-Pack M, et al.
Identification of proliferating dendritic cell precursors in mouse blood.
J Exp Med.
1992;175:1157-1165 34. Markowicz S, Engleman EG. GM-CSF promotes differentiation and survival of human peripheral blood dendritic cells. J Clin Invest. 1990;85:955-961.
35.
Salomon B, Cohen JL, Masurier C, Klatzmann D.
Three populations of mouse lymph node dendritic cells with different orgins and dynamics.
J Immunol.
1998;160:708-717
36.
Anjuere F, Martin P, Ferrero I, et al.
Definition of dendritic cell subpopulations present in the spleen, Peyer's patches, lymph nodes, and skin of the mouse.
Blood.
1999;93:590-598
37.
De Smedt T, Pajak B, Muraille E, et al.
Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo.
J Exp Med.
1996;184:1413-1420
38.
Paquette RL, Hsu NC, Kiertscher SM, et al.
Interferon-
39.
Luft T, Pang KC, Thomas E, et al.
Type I IFNs enhance the terminal differentiation of dendritic cells.
J Immunol.
1998;161:1947-1953 40. Anjuère F, Martínez del Hoyo G, Martín P, Ardavín C. Langerhan cells acquire a CD8+ dendritic cell phenotype on maturation by CD40 ligation. J Leukoc Biol. 2000;67:206-209[Abstract].
41.
Cella M, Salio M, Sakakibara Y, Langen H, Julkunen I, Lanzavecchia A.
Maturation, activation, and protection of dendritic cells induced by double-stranded RNA.
J Exp Med.
1999;189:821-829
42.
Olweus J, BitMansour A, Warnke R, et al.
Dendritic cell ontogeny: a human cell lineage of myeloid orgin.
Proc Natl Acad Sci U S A.
1997;94:12551-12556 43. Wu L, Vremec D, Ardavin C, et al. Mouse thymus dendritic cells: kinetics of development and changes in surface markers during maturation. Eur J Immunol. 1995;25:418-429[Medline] [Order article via Infotrieve].
44.
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 45. Ardavin C, Wu L, Li C-L, Shortman K. Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Nature. 1993;362:761-763[Medline] [Order article via Infotrieve]. 46. Izon DJ, Jones LA, Eynon EE, Kruisbeek AM. A molecule expressed on accessory cells, activated T cells, and thymic epithelium is a marker and promoter of T cell activation. J Immunol. 1994;153:2939-2950[Abstract]. 47. Izon DJ, Oritani K, Hamel M, et al. Identification and functional analysis of Ly-6A/E as a thymic and bone marrow stromal antigen. J Immunol. 1996;156:2391-2399[Abstract].
48.
Radtke F, Ferrero I, Wilson A, Lees R, Aguet M, MacDonald HR.
Notch1 deficiency dissociates the intrathymic development of dendritic cells and T cells.
J Exp Med.
2000;191:1085-1093
49.
Rodewald H-R, Brocker T, Haller C.
Developmental dissociation of thymic dendritic cell and thymocyte lineages revealed in growth factor receptor mutant mice.
Proc Natl Acad Sci U S A.
1999;96:15068-15073
© 2000 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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|
 |
|
  P. J. Dunne, B. Moran, R. C. Cummins, and K. H. G. Mills CD11c+CD8{alpha}+ Dendritic Cells Promote Protective Immunity to Respiratory Infection with Bordetella pertussis J. Immunol., July 1, 2009; 183(1): 400 - 410. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. K. Swee, N. Bosco, B. Malissen, R. Ceredig, and A. Rolink Expansion of peripheral naturally occurring T regulatory cells by Fms-like tyrosine kinase 3 ligand treatment Blood, June 18, 2009; 113(25): 6277 - 6287. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Merad and M. G. Manz Dendritic cell homeostasis Blood, April 9, 2009; 113(15): 3418 - 3427. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Cohen, G. K. Koski, B. J. Czerniecki, K. D. Bunting, X.-Y. Fu, Z. Wang, W.-J. Zhang, C. S. Carter, M. Awad, C. A. Distel, et al. STAT3- and STAT5-dependent pathways competitively regulate the pan-differentiation of CD34pos cells into tumor-competent dendritic cells Blood, September 1, 2008; 112(5): 1832 - 1843. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-C. Sheng, M. Kalkanidis, D. S. Pouniotis, M. D. Wright, G. A. Pietersz, and V. Apostolopoulos The Adjuvanticity of a Mannosylated Antigen Reveals TLR4 Functionality Essential for Subset Specialization and Functional Maturation of Mouse Dendritic Cells J. Immunol., August 15, 2008; 181(4): 2455 - 2464. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. V. Baev, S. Caielli, F. Ronchi, M. Coccia, F. Facciotti, K. E. Nichols, and M. Falcone Impaired SLAM-SLAM Homotypic Interaction between Invariant NKT Cells and Dendritic Cells Affects Differentiation of IL-4/IL-10-Secreting NKT2 Cells in Nonobese Diabetic Mice J. Immunol., July 15, 2008; 181(2): 869 - 877. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Schaefer, N. Reiling, C. Fessler, J. Stephani, I. Taniuchi, F. Hatam, A. O. Yildirim, H. Fehrenbach, K. Walter, J. Ruland, et al. Decreased Pathology and Prolonged Survival of Human DC-SIGN Transgenic Mice during Mycobacterial Infection J. Immunol., May 15, 2008; 180(10): 6836 - 6845. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Gautier, T. Huby, F. Saint-Charles, B. Ouzilleau, M. J. Chapman, and P. Lesnik Enhanced Dendritic Cell Survival Attenuates Lipopolysaccharide-Induced Immunosuppression and Increases Resistance to Lethal Endotoxic Shock J. Immunol., May 15, 2008; 180(10): 6941 - 6946. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Fancke, M. Suter, H. Hochrein, and M. O'Keeffe M-CSF: a novel plasmacytoid and conventional dendritic cell poietin Blood, January 1, 2008; 111(1): 150 - 159. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. L. Papenfuss, A. P. Kithcart, N. D. Powell, M. A. McClain, I. E. Gienapp, T. M. Shawler, and C. C. Whitacre Disease-modifying capability of murine Flt3-ligand DCs in experimental autoimmune encephalomyelitis J. Leukoc. Biol., December 1, 2007; 82(6): 1510 - 1518. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Xu, Y. Zhan, A. M. Lew, S. H. Naik, and M. H. Kershaw Differential Development of Murine Dendritic Cells by GM-CSF versus Flt3 Ligand Has Implications for Inflammation and Trafficking J. Immunol., December 1, 2007; 179(11): 7577 - 7584. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. H. Tan and H. C. O'Neill Concise Review: Dendritic Cell Development in the Context of the Spleen Microenvironment Stem Cells, September 1, 2007; 25(9): 2139 - 2145. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Meissner, M. Rutkowski, A. L. Harmsen, S. Han, and A. G. Harmsen Type I Interferon Signaling and B Cells Maintain Hemopoiesis during Pneumocystis Infection of the Lung J. Immunol., May 15, 2007; 178(10): 6604 - 6615. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Allman, M. Dalod, C. Asselin-Paturel, T. Delale, S. H. Robbins, G. Trinchieri, C. A. Biron, P. Kastner, and S. Chan Ikaros is required for plasmacytoid dendritic cell differentiation Blood, December 15, 2006; 108(13): 4025 - 4034. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Gabriele, A. Fragale, P. Borghi, P. Sestili, E. Stellacci, M. Venditti, G. Schiavoni, M. Sanchez, F. Belardelli, and A. Battistini IRF-1 deficiency skews the differentiation of dendritic cells toward plasmacytoid and tolerogenic features J. Leukoc. Biol., December 1, 2006; 80(6): 1500 - 1511. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wang, J. Arp, X. Huang, W. Liu, S. Ramcharran, J. Jiang, B. Garcia, N. Kanai, W. Min, P. J. O'Connell, et al. Distinct Subsets of Dendritic Cells Regulate the Pattern of Acute Xenograft Rejection and Susceptibility to Cyclosporine Therapy J. Immunol., March 15, 2006; 176(6): 3525 - 3535. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. D. Pawar, P. S. Patole, D. Zecher, S. Segerer, M. Kretzler, D. Schlondorff, and H.-J. Anders Toll-Like Receptor-7 Modulates Immune Complex Glomerulonephritis J. Am. Soc. Nephrol., January 1, 2006; 17(1): 141 - 149. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Tussiwand, N. Onai, L. Mazzucchelli, and M. G. Manz Inhibition of Natural Type I IFN-Producing and Dendritic Cell Development by a Small Molecule Receptor Tyrosine Kinase Inhibitor with Flt3 Affinity J. Immunol., September 15, 2005; 175(6): 3674 - 3680. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. G. Diacovo, A. L. Blasius, T. W. Mak, M. Cella, and M. Colonna Adhesive mechanisms governing interferon-producing cell recruitment into lymph nodes J. Exp. Med., September 6, 2005; 202(5): 687 - 696. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Minami, Y. Yanagawa, K. Iwabuchi, N. Shinohara, T. Harabayashi, K. Nonomura, and K. Onoe Negative feedback regulation of T helper type 1 (Th1)/Th2 cytokine balance via dendritic cell and natural killer T cell interactions Blood, September 1, 2005; 106(5): 1685 - 1693. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wakui, L. Morel, E. J. Butfiloski, C. Kim, and E. S. Sobel Genetic Dissection of Systemic Lupus Erythematosus Pathogenesis: Partial Functional Complementation between Sle1 and Sle3/5 Demonstrates Requirement for Intracellular Coexpression for Full Phenotypic Expression of Lupus J. Immunol., July 15, 2005; 175(2): 1337 - 1345. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. S. Angelov, M. Tomkowiak, A. Marcais, Y. Leverrier, and J. Marvel Flt3 Ligand-Generated Murine Plasmacytoid and Conventional Dendritic Cells Differ in Their Capacity to Prime Naive CD8 T Cells and to Generate Memory Cells In Vivo J. Immunol., July 1, 2005; 175(1): 189 - 195. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Galibert, G. S. Diemer, Z. Liu, R. S. Johnson, J. L. Smith, T. Walzer, M. R. Comeau, C. T. Rauch, M. F. Wolfson, R. A. Sorensen, et al. Nectin-like Protein 2 Defines a Subset of T-cell Zone Dendritic Cells and Is a Ligand for Class-I-restricted T-cell-associated Molecule J. Biol. Chem., June 10, 2005; 280(23): 21955 - 21964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. H. Naik, A. I. Proietto, N. S. Wilson, A. Dakic, P. Schnorrer, M. Fuchsberger, M. H. Lahoud, M. O'Keeffe, Q.-x. Shao, W.-f. Chen, et al. Cutting Edge: Generation of Splenic CD8+ and CD8- Dendritic Cell Equivalents in Fms-Like Tyrosine Kinase 3 Ligand Bone Marrow Cultures J. Immunol., June 1, 2005; 174(11): 6592 - 6597. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. S. Patole, H.-J. Grone, S. Segerer, R. Ciubar, E. Belemezova, A. Henger, M. Kretzler, D. Schlondorff, and H.-J. Anders Viral Double-Stranded RNA Aggravates Lupus Nephritis through Toll-Like Receptor 3 on Glomerular Mesangial Cells and Antigen-Presenting Cells J. Am. Soc. Nephrol., May 1, 2005; 16(5): 1326 - 1338. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hieronymus, T. C. Gust, R. D. Kirsch, T. Jorgas, G. Blendinger, M. Goncharenko, K. Supplitt, S. Rose-John, A. M. Muller, and M. Zenke Progressive and Controlled Development of Mouse Dendritic Cells from Flt3+CD11b+ Progenitors In Vitro J. Immunol., March 1, 2005; 174(5): 2552 - 2562. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. McKenna, A.-S. Beignon, and N. Bhardwaj Plasmacytoid Dendritic Cells: Linking Innate and Adaptive Immunity J. Virol., January 1, 2005; 79(1): 17 - 27. [Full Text] [PDF]
|
|
|
|
|
|
T. Walzer, L. Galibert, M. R. Comeau, and T. De Smedt Plexin C1 Engagement on Mouse Dendritic Cells by Viral Semaphorin A39R Induces Actin Cytoskeleton Rearrangement and Inhibits Integrin-Mediated Adhesion and Chemokine-Induced Migration J. Immunol., January 1, 2005; 174(1): 51 - 59. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. Hengesbach and K. A. Hoag Physiological Concentrations of Retinoic Acid Favor Myeloid Dendritic Cell Development over Granulocyte Development in Cultures of Bone Marrow Cells from Mice J. Nutr., October 1, 2004; 134(10): 2653 - 2659. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Andoniou, D. M. Andrews, M. Manzur, P. Ricciardi-Castagnoli, and M. A. Degli-Esposti A novel checkpoint in the Bcl-2-regulated apoptotic pathway revealed by murine cytomegalovirus infection of dendritic cells J. Cell Biol., September 13, 2004; 166(6): 827 - 837. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Suzuki, K. Honma, T. Matsuyama, K. Suzuki, K. Toriyama, I. Akitoyo, K. Yamamoto, T. Suematsu, M. Nakamura, K. Yui, et al. From the Cover: Critical roles of interferon regulatory factor 4 in CD11bhighCD8{alpha}- dendritic cell development PNAS, June 15, 2004; 101(24): 8981 - 8986. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. I. Bleier, V. G. Pillarisetty, A. B. Shah, and R. P. DeMatteo Increased and Long-Term Generation of Dendritic Cells with Reduced Function from IL-6-Deficient Bone Marrow J. Immunol., June 15, 2004; 172(12): 7408 - 7416. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Mohty, E. Jourdan, N. B. Mami, N. Vey, G. Damaj, D. Blaise, D. Isnardon, D. Olive, and B. Gaugler Imatinib and plasmacytoid dendritic cell function in patients with chronic myeloid leukemia Blood, June 15, 2004; 103(12): 4666 - 4668. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Blasius, W. Vermi, A. Krug, F. Facchetti, M. Cella, and M. Colonna A cell-surface molecule selectively expressed on murine natural interferon-producing cells that blocks secretion of interferon-alpha Blood, June 1, 2004; 103(11): 4201 - 4206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. C. O'Neill and H. L. Wilson Limitations with in vitro production of dendritic cells using cytokines J. Leukoc. Biol., April 1, 2004; 75(4): 600 - 603. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. C. Alaniz, S. Sandall, E. K. Thomas, and C. B. Wilson Increased Dendritic Cell Numbers Impair Protective Immunity to Intracellular Bacteria Despite Augmenting Antigen-Specific CD8+ T Lymphocyte Responses J. Immunol., March 15, 2004; 172(6): 3725 - 3735. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Honda, T. Mizutani, and T. Taniguchi Negative regulation of IFN-{alpha}/{beta} signaling by IFN regulatory factor 2 for homeostatic development of dendritic cells PNAS, February 24, 2004; 101(8): 2416 - 2421. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. H. Jackson, C.-R. Yu, R. M. Mahdi, S. Ebong, and C. E. Egwuagu Dendritic Cell Maturation Requires STAT1 and Is under Feedback Regulation by Suppressors of Cytokine Signaling J. Immunol., February 15, 2004; 172(4): 2307 - 2315. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Cheng, Y. Nefedova, L. Miele, B. A. Osborne, and D. Gabrilovich Notch signaling is necessary but not sufficient for differentiation of dendritic cells Blood, December 1, 2003; 102(12): 3980 - 3988. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. T. H. Coates, S. M. Barratt-Boyes, L. Zhang, V. S. Donnenberg, P. J. O'Connell, A. J. Logar, F. J. Duncan, M. Murphey-Corb, A. D. Donnenberg, A. E. Morelli, et al. Dendritic cell subsets in blood and lymphoid tissue of rhesus monkeys and their mobilization with Flt3 ligand Blood, October 1, 2003; 102(7): 2513 - 2521. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. D'Amico and L. Wu The Early Progenitors of Mouse Dendritic Cells and Plasmacytoid Predendritic Cells Are within the Bone Marrow Hemopoietic Precursors Expressing Flt3 J. Exp. Med., July 21, 2003; 198(2): 293 - 303. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Karsunky, M. Merad, A. Cozzio, I. L. Weissman, and M. G. Manz Flt3 Ligand Regulates Dendritic Cell Development from Flt3+ Lymphoid and Myeloid-committed Progenitors to Flt3+ Dendritic Cells In Vivo J. Exp. Med., July 21, 2003; 198(2): 305 - 313. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Nikolic, M. F. T. R. d. Bruijn, M. B. Lutz, and P. J. M. Leenen Developmental stages of myeloid dendritic cells in mouse bone marrow Int. Immunol., April 1, 2003; 15(4): 515 - 524. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Huber, D. Sartini, and M. Exley Role of CD1d in Coxsackievirus B3-Induced Myocarditis J. Immunol., March 15, 2003; 170(6): 3147 - 3153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Tsujimura, T. Tamura, C. Gongora, J. Aliberti, C. Reis e Sousa, A. Sher, and K. Ozato ICSBP/IRF-8 retrovirus transduction rescues dendritic cell development in vitro Blood, February 1, 2003; 101(3): 961 - 969. [Abstract] [Full Text] [PDF]
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J. Aliberti, O. Schulz, D. J. Pennington, H. Tsujimura, C. R. e Sousa, K. Ozato, and A. Sher Essential role for ICSBP in the in vivo development of murine CD8alpha + dendritic cells Blood, January 1, 2003; 101(1): 305 - 310. [Abstract] [Full Text] [PDF]
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P. Brawand, D. R. Fitzpatrick, B. W. Greenfield, K. Brasel, C. R. Maliszewski, and T. De Smedt Murine Plasmacytoid Pre-Dendritic Cells Generated from Flt3 Ligand-Supplemented Bone Marrow Cultures Are Immature APCs J. Immunol., December 15, 2002; 169(12): 6711 - 6719. [Abstract] [Full Text] [PDF]
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T. Nakajima, N. Inagaki, H. Tanaka, A. Tanaka, M. Yoshikawa, M. Tamari, K. Hasegawa, K. Matsumoto, H. Tachimoto, M. Ebisawa, et al. Marked increase in CC chemokine gene expression in both human and mouse mast cell transcriptomes following Fcepsilon receptor I cross-linking: an interspecies comparison Blood, December 1, 2002; 100(12): 3861 - 3868. [Abstract] [Full Text] [PDF]
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B. J. Weigel, N. Nath, P. A. Taylor, A. Panoskaltsis-Mortari, W. Chen, A. M. Krieg, K. Brasel, and B. R. Blazar Comparative analysis of murine marrow-derived dendritic cells generated by Flt3L or GM-CSF/IL-4 and matured with immune stimulatory agents on the in vivo induction of antileukemia responses Blood, December 1, 2002; 100(12): 4169 - 4176. [Abstract] [Full Text] [PDF]
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M. O'Keeffe, H. Hochrein, D. Vremec, I. Caminschi, J. L. Miller, E. M. Anders, L. Wu, M. H. Lahoud, S. Henri, B. Scott, et al. Mouse Plasmacytoid Cells: Long-lived Cells, Heterogeneous in Surface Phenotype and Function, that Differentiate Into CD8+ Dendritic Cells Only after Microbial Stimulus J. Exp. Med., November 18, 2002; 196(10): 1307 - 1319. [Abstract] [Full Text] [PDF]
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L. S. van Rijt, J.-B. Prins, P. J. M. Leenen, K. Thielemans, V. C. de Vries, H. C. Hoogsteden, and B. N. Lambrecht Allergen-induced accumulation of airway dendritic cells is supported by an increase in CD31hiLy-6Cneg bone marrow precursors in a mouse model of asthma Blood, November 15, 2002; 100(10): 3663 - 3671. [Abstract] [Full Text] [PDF]
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R. J. Steptoe, J. M. Ritchie, and L. C. Harrison Increased Generation of Dendritic Cells from Myeloid Progenitors in Autoimmune-Prone Nonobese Diabetic Mice J. Immunol., May 15, 2002; 168(10): 5032 - 5041. [Abstract] [Full Text] [PDF]
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Y. Valdez, W. Mah, M. M. Winslow, L. Xu, P. Ling, and S. E. Townsend Major Histocompatibility Complex Class II Presentation of Cell-associated Antigen Is Mediated by CD8{alpha}+ Dendritic Cells In Vivo J. Exp. Med., March 11, 2002; 195(6): 683 - 694. [Abstract] [Full Text] [PDF]
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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; 167(2): 741 - 748. [Abstract] [Full Text] [PDF]
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M. Gilliet, A. Boonstra, C. Paturel, S. Antonenko, X.-L. Xu, G. Trinchieri, A. O'Garra, and Y.-J. Liu The Development of Murine Plasmacytoid Dendritic Cell Precursors Is Differentially Regulated by FLT3-ligand and Granulocyte/Macrophage Colony-Stimulating Factor J. Exp. Med., April 1, 2002; 195(7): 953 - 958. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) and "lymphoid-related"
(CD11bdull, CD8
), 100 ng/mL of FL (
), or 100 ng/mL
SCF (
) for 10 days and then harvested from the lower chamber,
counted, and analyzed for expression of cell-surface antigens by flow
cytometry. Results shown are representative of 3 experiments.
), CD11c (
) and BM-derived DCs from cultures
using GM-CSF and IL-4 (
0.044 ± 0.031. Background OD from DCs alone was
subtracted from DCs with T cells.
