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Flt3-Ligand and Granulocyte
Colony-Stimulating Factor Mobilize Distinct
Human Dendritic Cell Subsets In Vivo
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Bali Pulendran, Jacques Banchereau, Susan Burkeholder,
Elizabeth Kraus, Elisabeth Guinet, Cecile Chalouni, Dania
Caron, Charles Maliszewski, Jean Davoust, Joseph Fay and
Karolina Palucka
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2000 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2000; 165:566-572; ;
doi: 10.4049/jimmunol.165.1.566
http://www.jimmunol.org/content/165/1/566
Flt3-Ligand and Granulocyte Colony-Stimulating Factor
Mobilize Distinct Human Dendritic Cell Subsets In Vivo1
Bali Pulendran,2* Jacques Banchereau,* Susan Burkeholder,* Elizabeth Kraus,*
Elisabeth Guinet,* Cecile Chalouni,* Dania Caron,† Charles Maliszewski,† Jean Davoust,*
Joseph Fay,* and Karolina Palucka*
D
endritic cells (DCs)3 constitute a system of rare APCs
that play crucial roles in the elicitation of T cell-dependent immunity (1, 2). The development of DCs is considered to occur in distinct stages. Proliferating DC progenitors in
the bone marrow develop into DC precursors, which circulate in
the blood. These give rise to immature DCs, which are strategically positioned in various nonlymphoid tissues in the body where
they can capture and process Ags from invading pathogens. Proinflammatory signals that are often triggered by infectious agents
initiate the maturation of these DCs and their migration to the T
cell-rich areas of the lymph nodes and spleen, where they present
captured Ags to naive T cells (1, 2). Ag-specific clonal expansion
is initiated, and this eventually leads to elimination of the pathogen
and establishment of immunological memory.
Although it is known that DCs are critical in initiating T cell
immunity, emerging evidence suggests that DCs also play roles in
the regulation of such responses. For example, distinct DC subsets
can differentially regulate the Th1/Th2 balance in vivo (3, 4) and
in vitro (5). Much information about DCs has accrued from the
study of DCs grown in vitro under the influence of cytokines such
*Baylor Institute for Immunology Research, Dallas, TX 75204; and †Immunex Corporation, Seattle, WA 98101
Received for publication December 28, 1999. Accepted for publication April
17, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Baylor Health Care System Foundation
and the National Institutes of Health (CA78846-01A1).
2
Address correspondence and reprint requests to Dr. Bali Pulendran, Baylor Institute
for Immunology Research, 3434 Live Oak, Dallas, TX 75204. E-mail address:
[email protected]
3
Abbreviations used in this paper: DC, dendritic cell; FL, Flt3-ligand; CD40L, CD40
ligand; LAMP, lysosome-associated membrane protein.
Copyright © 2000 by The American Association of Immunologists
as GM-CSF (6 –9). However the study of DCs in vivo has been
difficult because of their rarity in the blood and in other tissues. In
mice, the identification of cytokines such as Flt3-ligand (FL) that
mobilize DCs in vivo has offered an attractive means of expanding
various DC subsets in vivo (3, 10 –12). FL has been shown to
expand distinct DC subsets in mice and to greatly augment Agspecific T and B cell responses against soluble Ags and tumors (3,
13, 14).
In humans, cytokines such as GM-CSF and FL play crucial roles
in the expansion and maturation of DCs in vitro. Therefore, investigating the effects of such cytokines on DC function in vivo is
of paramount importance, especially from a clinical perspective. In
particular, it is essential to determine whether distinct cytokines
might elicit the expansion of functionally different DC subsets in
humans, as is seen in mice (3). If indeed they do, this may open up
new possibilities for immunomodulation and may offer more
mechanistic and rational approaches to the use of such cytokines in
enhancing anti-infectious or antitumor immunity. In this context,
the immunomodulatory effects of cytokines such as GM-CSF and
G-CSF are well-known. For example, GM-CSF has been used as
an effective vaccine adjuvant for protein- and peptide-based vaccines (15, 16). G-CSF, despite mobilizing large numbers of mononuclear cells, does not increase the severity of acute graft-versushost disease after allogeneic bone marrow transplantion compared
with historical control bone marrow grafts (17–19). In addition,
G-CSF suppresses T cell proliferation (20 –22) and the generation
of cytolytic effectors (22). However, the immunological mechanisms underlying such potent effects are unknown. Because DCs
play vital roles in the regulation of immune responses (1–5), it is
possible that these cytokines may act through the generation of
functionally distinct DC populations in vivo. To this end, we are
investigating the effects of cytokines on in vivo DC mobilization in
healthy volunteers.
0022-1767/00/$02.00
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Dendritic cells (DCs) have a unique ability to stimulate naive T cells. Recent evidence suggests that distinct DC subsets direct
different classes of immune responses in vitro and in vivo. In humans, the monocyte-derived CD11cⴙ DCs induce T cells to produce
Th1 cytokines in vitro, whereas the CD11cⴚ plasmacytoid T cell-derived DCs elicit the production of Th2 cytokines. In this paper
we report that administration of either Flt3-ligand (FL) or G-CSF to healthy human volunteers dramatically increases distinct DC
subsets, or DC precursors, in the blood. FL increases both the CD11cⴙ DC subset (48-fold) and the CD11cⴚ IL-3Rⴙ DC precursors
(13-fold). In contrast, G-CSF only increases the CD11cⴚ precursors (>7-fold). Freshly sorted CD11cⴙ but not CD11cⴚ cells
stimulate CD4ⴙ T cells in an allogeneic MLR, whereas only the CD11cⴚ cells can be induced to secrete high levels of IFN-␣, in
response to influenza virus. CD11cⴙ and CD11cⴚ cells can mature in vitro with GM-CSF ⴙ TNF-␣ or with IL-3 ⴙ CD40 ligand,
respectively. These two subsets up-regulate MHC class II costimulatory molecules as well as the DC maturation marker DClysosome-associated membrane protein, and they stimulate naive, allogeneic CD4ⴙ T cells efficiently. These two DC subsets elicit
distinct cytokine profiles in CD4ⴙ T cells, with the CD11cⴚ subset inducing higher levels of the Th2 cytokine IL-10. The differential
mobilization of distinct DC subsets or DC precursors by in vivo administration of FL and G-CSF offers a novel strategy to
manipulate immune responses in humans. The Journal of Immunology, 2000, 165: 566 –572.
The Journal of Immunology
We report in this paper that FL and G-CSF dramatically enhance
the numbers of distinct DC subsets in the peripheral blood of
healthy human volunteers. In particular, FL expands two subsets of
precursor or immature DCs that circulate in human blood and that
can be identified by the differential expression of CD11c, the immature CD11c⫹ DC subset, and the precursor CD11c⫺ DC subset
(1, 5, 23–28). In contrast, G-CSF results in a preferential expansion of the CD11c⫺ subset.
Materials and Methods
Patient selection criteria
Abs and reagents
Purified mAbs used in this study were: CD11b, CD11c, CD14, CD2, CD5,
CD8, CD80, CD69, CD16, CD123, HLA-DR (BD-PharMingen, San Diego); CD13, CD33, CD86, CD95, CD116, CD55, CD59 (PharMingen, San
Diego, CA); CD4, CD1a, CD83, CD40, CD40 ligand (CD40L), CD54,
CD135, CD12 (Coulter/Immunotech, Palo Alto, CA); and CD32 (Caltag,
South San Francisco, CA). Recombinant human cytokines used in this
study were: rhGM-CSF (Immunex; final concentration of 100 ng/ml);
TNF-␣ (Boehringer Ingelheim, Ridgefield, CT; final concentration of 10
ng/ml); IL-3 (Immunex; final concentration of 10 ng/ml); and CD40L (Immunex; final concentration of 200 ng/ml). RPMI complete (RPMIc) medium consisted of RPMI 1640, 1% L-glutamine, 1% penicillin/streptomycin, 50 mM 2-ME, 1% sodium pyruvate, 1% essential amino acids, and
heat-inactivated 10% FCS (all from Life Technologies, Grand Island, NY).
Flow cytometric identification and purification of CD11c⫹ and
CD11c⫺ DCs
CD11c⫹ and CD11c⫺ blood DCs were isolated from mononuclear fraction
according to the study protocol. The mononuclear cell fractions from the
apheresed samples were run over a CD34 column (Cell Pro, Seattle, WA)
to deplete CD34⫹ cells. Briefly, DC precursors were enriched from mononuclear cells by magnetic bead depletion (goat anti-mouse IgG Dynabeads
from Dynal, Lake Success, NY) after incubation with a cocktail of mAbs
to lineage markers including CD3, CD14, CD16, CD19, CD56, and glycophorin A (Coulter, Palo Alto, CA). The recovery after depletion ranged from 6
to 19% at day 0 and from 11 to 46% after FL treatment (day 10). The recovered DC fraction was labeled using FITC-lineage cocktail, APC-CD11c, peridinin chlorophyl protein-HLA-DR, and PE-CD123 and was sorted as LINneg,
HLA-DR⫹CD11c⫹ and HLA-DR⫹CD11c⫺CD123⫹ populations. Sorting
was accomplished on a FACSVantage flow cytometer (Becton Dickinson)
equipped with an Enterprise II laser (Coherent Radiatin, Palo Alto, CA).
Cell culture of sorted CD11c⫹ and CD11c⫺ populations
Sorted CD11c⫹ and CD11c⫺ cells were cultured at 1 ⫻ 106 cells/ml in
RPMIc ⫹ 5% FBS and were cultured in the presence of GM-CSF ⫹
TNF-␣ or IL-3 ⫹ CD40L, respectively, for 5 days.
T cell proliferation and cytokine assay
Freshly isolated CD11c⫹ and CD11c⫺ cells, or in vitro-matured DCs,
were cultured with freshly isolated CD4⫹, CD45RA⫹ allogeneic T cells
from cord blood or from adult blood. Allogeneic T cells were purified
by anti-CD8/CD16/CD19/CD56/CD14/HLA-DR/CD45RO-based immunomagnetic depletion of PBMCs from adult blood or cord blood. DC
T cell cultures were set up for 5 days in RPMIc ⫹ 5% FBS. Cells were
pulsed for the last 10 h with 1.0 ␮Ci [3H]thymidine per well, and incorporation of the radionucleotide was measured by ␤-scintillation
spectroscopy. For cytokine analysis, supernatants were harvested 5 days
after culture, and the cells were restimulated with PHA in fresh medium
for 24 h. Cytokines released were assayed by ELISA kits from R&D
Systems (Minneapolis, MN).
Confocal microscopy
Intracellular immunofluorescence staining was performed as previously described for suspension cells (29). Briefly, the cells were allowed to adhere
on polylysin-coated coverslips for 1 h at room temperature, fixed for 15
min with 4% paraformaldehyde in PBS, permeabilized and labeled with
anti-DC-lysosome-associated membrane protein (LAMP) (generous gift
from Dr. S. Lebecque, Schering-Plough, Dardilly, France), revealed with
donkey anti-mouse Abs coupled to Texas Red (Jackson Laboratories, West
Grove, PA), and/or labeled with anti-HLA-DR, anti-CD1a coupled to FITC
(Becton Dickinson). Coverslips were mounted onto glass slides with
Fluoromount (Southern Biotechnology Associates, Birmingham, AL).
Confocal microscopy was performed using a TCS SP microscope equipped
with argon and krypton ion lasers and a ⫻100 1.4 NA PLAPO objective
(Leica Microsystem, Heidelberg, Germany).
IFN-␣ assay
Freshly sorted CD11c⫹, CD11c⫺, and CD14⫹CD11c⫹ cells (monocytes)
were cultured in RPMIc ⫹ 5% FCS at 2 ⫻ 105 cells/ml with various doses
of influenza virus for 24 h. Supernatants were harvested and assayed for
IFN-␣ using an ELISA assay kit from BioSource (Camarillo, CA).
Results and Discussion
FL mobilizes both CD11c⫹ DCs and CD11c⫺ pre-DCs in vivo
Six healthy adult volunteers were injected with FL s.c. at 10 ␮g/
kg/day for 10 days. Previous studies in mice (10, 11) and recent
work in humans (26) suggest that optimal DC expansion is seen
after 10 days of FL injections. The volunteers were apheresed at
days 0 and 10 to collect mononuclear cells. The mononuclear fractions from FL-treated volunteers were analyzed by flow cytometry
to assess the frequencies and absolute numbers of various APCs in
the blood. The mobilization of the CD11c⫹ and CD11c⫺ populations at day 10 was evaluated. The CD11c⫹ and CD11c⫺ subsets
were defined by lack of lineage marker expression
(CD3⫺CD14⫺CD16⫺CD19⫺CD56⫺), expression of HLA-DR,
and differential expression of CD11c and CD123 (Fig. 1). In absolute numbers per milliliter of blood, the CD11c⫹ cells are increased 48-fold, from 36,354 ⫾ 6333 per ml (n ⫽ 5; range,
23,520 –57,850) to 1,759,423 ⫾ 547,215 per ml (n ⫽ 5; range,
867,970 –3,489,600) (Fig. 2A). In contrast, the number of CD11c⫺
DCs are increased 13-fold, from 28,880 ⫾ 11,764 per ml (n ⫽ 5;
range, 12,200 – 49,400) to 387,300 ⫾ 93,112 per ml (n ⫽ 5; range,
232,100 – 681,600) (Fig. 2B). Thus, the numeric ratio of CD11c⫹/
CD11c⫺ DCs is considerably increased in FL-treated donors compared with baseline controls (Fig. 2C).
Wright Giemsa staining of sorted CD11c⫹ cells revealed a distinctive, multilobulated nuclear morphology, and few of these cells
expressed dendrites (data not shown). The CD11c⫺ cells showed a
typical plasmacytoid-like morphology, characterized by a large,
eccentric nucleus with a granular cytoplasm (data not shown).
G-CSF treatment preferentially expands the CD11c⫺ pre-DC
subset in peripheral blood
Five healthy adult volunteers were treated with G-CSF s.c. at 10
␮g/kg/day for 5 days and were apheresed at day 6. The mononuclear cell fractions from these volunteers were analyzed by flow
cytometry after lineage depletion in the same manner as FL samples were. G-CSF preferentially increases the CD11c⫺ pre-DC
population (Figs. 1 and 2). This increase is reflected in an increase
in the absolute numbers of CD11c⫺ pre-DCs (Fig. 2B) from
36,354 per ml at day 0 to 205,786 ⫾ 67,876 per ml (n ⫽ 5; range,
44,928 –500,610) at day 5. In contrast, the CD11c⫹ DCs are unchanged in their absolute numbers. Thus G-CSF, in contrast to FL,
preferentially mobilizes the CD11c⫺ pre-DC subset (Fig. 2, B and C).
The morphology of CD11c⫹ and CD11c⫺ cells from G-CSFtreated volunteers was similar to the equivalent cells from FLtreated individuals. The CD11c⫹ DCs had multilobulated nuclei
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In this clinical study, a total of 12 healthy volunteers received either FL (Immunex, Seattle, WA) s.c. at 10 ␮g/kg/day for 10 days (six volunteers; institutional review board no. 098-024) or G-CSF s.c. at 10 ␮g/kg/day for 5 days
(six volunteers; institutional review board no. 097-053). The volunteers had
HLA-A2 phenotype and normal blood counts and chemistries, and they were
⬎21 years of age. Informed written consent was obtained from each of the
volunteers. FL-treated volunteers underwent apheresis at baseline and after 10
days of FL administration to collect large numbers of cells for immunological
studies. G-CSF-treated volunteers underwent apheresis after 5 days of G-CSF
administration (i.e., at day 6). Both FL and G-CSF were well-tolerated by the
volunteers.
567
568
MOBILIZATION OF HUMAN DC SUBSETS BY Flt3-LIGAND AND G-CSF
FIGURE 1. Expansion of distinct DC
and pre-DC subsets by FL and G-CSF.
Flow cytometric identification of
CD11c⫹ DC and CD11c⫺ pre-DC subsets in FL and G-CSF donors. PBMCs
from FL-treated donors (day 10) or GCSF-treated donors (day 5) were gated
on forward and side light scatter (top left)
and were excluded of all lineage-positive
cells (CD3, CD14, CD16, CD19, and
CD56; bottom left). The lineage-negative
cells were analyzed for the expression of
CD11c, CD123, and HLA-DR. FL donors had elevated levels of both CD11c⫹
and CD11c⫺ cells compared with baseline controls, whereas G-CSF donors had a
preferential expansion of CD11c⫺ cells.
CD11c⫹ and CD11c⫺ cells from FL- and G-CSF-treated donors
are similar to the corresponding cells in nontreated donors
An extensive phenotypic analysis of the CD11c⫹ and CD11c⫺
subsets from FL and G-CSF volunteers was performed. DC-enriched fractions from FL- or G-CSF-treated donors were stained
with a series of Abs (Table I). The CD11c⫹ and the CD11c⫺
subsets expanded by FL treatment were very similar to the corresponding subsets in G-CSF-treated volunteers. CD11c⫺ subsets
from both FL and G-CSF donors expressed high levels of IL-3R
(CD123; Table I), confirming the identity of this subset as the
plasmacytoid T cell-related DC precursors reported earlier (23). As
previously described (23–25), the CD11c⫹ and CD11c⫺ subsets
differed phenotypically with respect to several markers. The
CD11c⫺ pre-DC subset expresses low levels of the myeloid markers CD11b, CD13, and CD33. CD2, CD5, and CD4, which are
expressed on T cells, are also expressed by this subset. Cells in this
subset do not express CD80 and express low levels of CD40 and
negligible levels of CD86. Overall, based on their morphology and
on the low expression of costimulatory molecules, the CD11c⫺
cells do not appear to be mature DCs.
The CD11c⫹ subset from FL- and G-CSF-treated donors express higher levels of HLA-DR, as well as of CD86 and CD40.
The CD11c⫹ subset also expresses high levels of the myeloid
markers CD11b, CD13, and CD33. The CD11c⫹ subset from GCSF-treated donors appears to be very similar in phenotype to the
CD11c⫹ subset from FL volunteers.
FIGURE 2. FL expands both CD11c⫹
DCs and CD11c⫺ pre-DCs, but G-CSF
preferentially expands the CD11c⫺ preDCs. Absolute numbers of CD11c⫹ (A)
and CD11c⫺ (B) cells in the blood of FL
and G-CSF donors. The absolute numbers per milliliter of blood were obtained
by multiplying the percentage of
CD11c⫹ and CD11c⫺ cells (assessed by
flow cytometry) by the numbers of PBMCs per milliliter of blood. Data are
based on five FL-treated donors and five
G-CSF-treated donors. C, Ratio of the
absolute numbers per milliliter of blood
of CD11c⫹ and CD11c⫺ cells.
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and possessed very few dendrites, whereas the CD11c⫺ pre-DCs
had large eccentric nuclei with a granular cytoplasm (data not
shown).
The Journal of Immunology
569
Table I. Phenotype of CD11c⫹ and CD11c⫺ cells from FL and G-CSF treated donors.a
FLT3-L
CD11c
a
b
G-CSF
⫺
CD11c
CD11c
⫺
CD11c⫹
⫹⫹
⫹⫹⫹
⫺
⫹⫹⫹
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫹
⫹⫹
⫹⫹⫹
⫺
⫹⫹⫹
⫹⫹⫹
⫹/⫺
⫺
⫹⫹
⫺
⫺
⫹⫹
⫺
⫺
⫹⫹⫹
⫹/⫺
⫺
⫹⫹
⫺
⫺
⫹/⫺
⫹
⫺
⫹⫹
⫹
⫺
⫺
⫺
⫹/⫺
⫺
⫹⫹
⫹
⫺
⫺
⫺
⫹
⫺
⫹⫹
⫹⫹
⫺
⫺
⫹/⫺
⫹
⫺
⫹⫹
⫺
⫹
⫺
⫹/⫺
⫺
⫹/⫺
⫺
⫹
⫹/⫺
⫺
⫹/⫺
⫹⫹⫹
⫺
⫹/⫺
⫹⫹⫹
⫺
⫹/⫺
⫹/⫺
⫺
⫹/⫺
⫹⫹
⫹⫹
⫹⫹
⫹⫹⫹ and ⫹/⫺b
⫹
⫺
⫹⫹
⫹⫹
⫹⫹ and ⫹/⫺b
⫹⫹ and ⫹/⫺b
⫹⫹
⫺
⫹⫹
⫹⫹
⫹⫹⫹ and ⫹/⫺b
⫹⫹ and ⫹/⫺b
⫹⫹
⫺
⫹⫹
⫹⫹
⫹⫹
⫹⫹⫹ and ⫹/⫺b
⫹
⫺
⫹⫹⫹, 103–104; ⫹⫹, 102–103; ⫹, 101–102; ⫺, 100–101.
Bimodal (two subpopulations).
Allostimulatory capacities of freshly isolated CD11c⫹ and
CD11c⫺ cells
CD11c⫹ and CD11c⫺ cells from FL donors were sorted and assessed for their capacity to stimulate naive CD4⫹ T cells in an
allogeneic MLR reaction. The CD11c⫹ cells could efficiently stimulate naive CD4⫹ T cells, but the CD11c⫺ cells were unable to
stimulate naive CD4⫹ T cells (Fig. 3). Based on their higher allostimulatory capacity and on their higher level of expression of
surface HLA-DR, the CD11c⫹ cells appear to be more mature than
the CD11c⫺ cells. Therefore, the CD11c⫹ cells display some characteristics of immature DCs, whereas the CD11c⫺ subset likely
represents precursors of DCs. However, it is important to stress
that the CD11c⫹ population has the potential to develop into several different types of APCs under the appropriate stimuli. For
example, the CD11c⫹ population can be induced to differentiate
into macrophages by M-CSF (27) (K. Palucka, B. Pulendran, A.
Rolland, N. Taquet, E. Neidhart-Berard, P. Blanco, S. Burkeholder, E. Kraus, J. Daroust, C. Chalouui, J. Fay, C. Maliszewski,
and J. Baucherean, manuscript in preparation), into Langerhans
cells by TGF-␤ (28) (K. Palucka et al., manuscript in preparation),
or into myeloid DCs by GM-CSF ⫹ TNF-␣ (24, 25, 27). This
suggests that the CD11c⫹ population contains precursor cells that
can develop into the various types of mature cells under different
stimuli. Whether these various mature cell types develop from the
very same precursor cells or from distinct cells is presently under
investigation (K. Palucka et al., manuscript in preparation).
IFN-␣ production by freshly isolated CD11c⫹ and CD11c⫺
cells
FIGURE 3. Allostimulatory capacity of freshly isolated CD11c⫹ and
CD11c⫺ cells. Cells were sorted by flow cytometry from FL donors and
cultured with 105 naive (CD45RA⫹) allogeneic CD4⫹ T cells for 5 days.
Cultures were pulsed with thymidine for the last 10 h. Data are representative of four independent experiments.
Two recent reports suggest that CD11c⫺ pre-DCs, freshly isolated
(24) or cultured in vitro for 2 days with IL-3 (31), secrete large
amounts of IFN-␣ in response to viruses. In this study, we wished
to determine whether CD11c⫹ and CD11c⫺ mobilized in vivo by
cytokines would behave similarly. CD11c⫹ and CD11c⫺ cells
were sorted by flow cytomtery and cultured in vitro with various
doses of influenza virus for 24 h. Sorted CD14⫹CD11c⫹ monocytes were also cultured with influenza virus as a control. As
shown in Fig. 4, the CD11c⫺ cells secrete much higher levels of
IFN-␣ than CD14⫹CD11c⫹ monocytes do. The CD11c⫹ cells did
not secrete IFN-␣ at any of the doses tested. The reduction in
IFN-␣ levels observed at 1000 U/ml may reflect the lysis of cells
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Myeloid markers
CD11b
CD13
CD14
CD33
DC maturation markers
HLA-DR
CD1a
CD83
Activation markers
CD40
CD40L
CD80
CD86
CD95
CD69
CD54
Fc receptors
CD16
CD32
Cytokine receptors
CD123 (IL-3R)
CD135 (Flt-3R)
CD126 (IL-6R)
Miscellaneous
CD55
CD59
CD2
CD5
CD4
CD8␣␤
⫹
570
MOBILIZATION OF HUMAN DC SUBSETS BY Flt3-LIGAND AND G-CSF
by excessive infection. These data are consistent with previous
reports that CD11c⫺ cells secrete IFN-␣ in response to viruses.
In vitro maturation of CD11c⫹ and CD11c⫺ cells
We wished to determine whether the DC subsets mobilized by FL and
G-CSF treatment could be induced to undergo maturation in vitro.
Previous reports have demonstrated that CD11c⫹ and CD11c⫺ DCs
can be matured in vitro with GM ⫹ TNF-␣ and IL-3 ⫹ CD40L,
respectively (5, 24, 25). In this study, we sorted the CD11c⫹ and
CD11c⫺ DC subsets from FL and G-CSF donors and cultured them
in vitro with the respective cytokines. After 5 days, we assessed the
phenotype, morphology, and allostimulatory capacity of the mature
DCs. CD11c⫹ DCs cultured in vitro with GM ⫹ TNF-␣ up-regulated
HLA-DR (Fig. 5), CD86, and CD40, but down-regulated CD1a (data
not shown), suggesting maturation. Confocal microscopy of the cultured CD11c⫹ DCs revealed a classical DC morphology, with many
dendrites, abundant veils, and bright HLA-DR surface staining, suggesting translocation of HLA-DR from the internal compartments revealed in freshly isolated cells (Fig. 5).
FIGURE 5. In vitro maturation of CD11c⫹ DCs and CD11c⫺ pre-DCs. Expression of CD1a and HLA-DR in ex vivo-isolated (left and middle panels)
and in vitro-cultured (right panels) CD11c⫹ and CD11c⫺ DC subsets. Single-color confocal microscopy of CD1a (left panels) and HLA-DR (middle and
right panels) was performed on CD11c⫹ DCs (upper panels) and CD11c⫺ DCs (lower panels). After in vitro culture of CD11c⫹ and CD11c⫺ DCs for
5 days with GM-CSF plus TNF-␣ and IL-3 plus CD40L, respectively, both DC cell types display dendritic projections and high amounts of surface
HLA-DR. Field, 60 ⫻ 60 ␮m.
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FIGURE 4. IFN-␣ production by freshly isolated CD11c⫹ and CD11c⫺
DC subsets from FL-treated donors. CD11c⫹, CD11c⫺, and CD14⫹CD11c⫹
monocytes were isolated from FL-treated donors and cultured for 24 h with
various doses of influenza virus (HA). CD11c⫺ pre-DCs produce large
amounts of IFN-␣ in response to HA.
FIGURE 6. DC-LAMP expression in maturing CD11c⫹ and CD11c⫺
DCs. Two-color immunofluorescence confocal microscopy of HLA-DR
and DC-LAMP was performed on CD11c⫹ DCs (A and B) and CD11c⫺
DCs (C and D) cultured in vitro for 5 days with GM-CSF plus TNF-␣ and
IL-3 plus CD40L, respectively. HLA-DR expression is shown in A and C.
DC-LAMP expression in cells of the same fields as in A and C is shown in
B and D. Optical sections performed 1 ␮m above the cell support to reveal
the accumulation of intracellular DC-LAMP. Field, 70 ⫻ 60 ␮m.
The Journal of Immunology
571
The CD11c⫺ DCs cultured in vitro in IL-3 ⫹ CD40L up-regulated
HLA-DR, CD80, and CD86 significantly (Fig. 5 and data not shown).
Confocal staining of these cells revealed numerous dendrites and upregulation of surface HLA-DR (Fig. 5). Thus, IL-3 ⫹ CD40L appears
to induce a maturation of the CD11c⫺ DCs. DC-LAMP, which is
specifically induced in maturing DCs derived from monocytes and
cord blood (29), was also up-regulated on the CD11c⫹ and the
CD11c⫺ subsets after in vitro maturation (Fig. 6). Expression of DCLAMP was confined to intracellular compartments, which is consistent with its localization in lysosomes (29).
CD11c⫹ and CD11c⫺ cells were matured in vitro and cultured
with allogeneic naive CD4⫹ T cells. Both DCs could stimulate
naive CD4⫹ T cells efficiently, although the CD11c⫺ DCs were
weaker (Fig. 7A). The CD11c⫺ precursors from both FL and GCSF donors gave the same yield of DCs when cultured in vitro.
These CD11c⫺ DCs from both FL- and G-CSF-treated individuals
appear to be very similar with respect to their allostimulatory
capacities.
CD11c⫹ and CD11c⫺ DCs elicit distinct cytokine patterns in
T cells
It has been reported that CD11c⫹ and CD11c⫺ DCs differ in the
cytokine profiles they induce in T cells. One recent report suggests
that monocyte-derived DCs elicit a polarized Th1 response,
whereas CD11c⫺ DCs elicit a Th2 response (5). A second report
examined CD11c⫹ and CD11c⫺ DCs from the peripheral blood
for Th1/Th2 skewing but failed to detect strongly polarized Th1
and Th2 responses (24). The discrepancy between these studies
may reflect the fact that monocyte-derived DCs were used in the
study by Rissoan et al. (5), whereas DCs derived from CD11c⫹
cells were used in the later study (24). Alternatively, the discrepancy could be because of differences in the maturation stages of the
CD11c⫹ DCs used or because of differences in the cytokines used
to mature them in vitro.
Therefore, we assessed whether the CD11c⫹ and CD11c⫺ DCs
mobilized by FL could differentially skew cytokine production in
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FIGURE 7. CD11c⫹ and CD11c⫺ DCs elicit distinct cytokine profiles in naive, allogeneic CD4⫹ T cells. A, Allostimulatory capacity of CD11c⫹ and
CD11c⫺ DCs induced to undergo maturation in vitro. CD11c⫹ and CD11c⫺ cells were sorted from FL or G-CSF donors and cultured for 5 days with GM
⫹ TNF-␣ or with IL-3 ⫹ CD40L, respectively. Then the cells were washed and cultured with 105 naive (CD45RA⫹) allogeneic CD4⫹ T cells for 5 days.
Cultures were pulsed with thymidine for the last 10 h of culture. B, CD11c⫹ and CD11c⫺ DCs induce distinct cytokine profiles in naive allogeneic CD4⫹
T cells. CD11c⫹ and CD11c⫺ were induced to maturation as described above and were cultured with T cells (5000 DCs; 105 T cells). Five days later,
supernatants were harvested, and the cells were restimulated with PHA for 24 h. The supernatants were harvested and assayed for cytokines using ELISA.
Note that naive CD4⫹ T cells cultured in the absence of DCs do not secrete any IFN-␥, IL-4, or IL-10. The histograms represent the mean values from
seven independent experiments. Differences in IL-10 levels between CD11c⫹ and CD11c⫺ are highly significant (CD11c⫹ vs CD11c⫺ (FL), p ⬍ 0.006;
CD11c⫹ vs CD11c⫺ (G), p ⬍ 0.007). Differences in IFN-␥ levels between CD11c⫹ and CD11c⫺ are significant (CD11c⫹ vs CD11c⫺ (FL), p ⬍ 0.05;
CD11c⫹ vs CD11c⫺ (G), p ⬍ 0.05). Differences in IL-4 levels are not significant. Student’s t test was used for statistical analyses.
572
MOBILIZATION OF HUMAN DC SUBSETS BY Flt3-LIGAND AND G-CSF
Acknowledgments
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
We thank Dr. Victor Garcia and colleagues (University of Texas Southwestern, Dallas, TX) for supplying cord blood and Dr. Madhav Dhodapkar
(Rockefeller University, New York, NY) for providing influenza virus. The
anti-DC-LAMP was a generous gift from Dr. S. Lebecque (ScheringPlough, Dardilly, France).
24.
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T cells. CD11c⫹ and CD11c⫺ cells were sorted and cultured for 5
days with GM-CSF ⫹ TNF-␣ and with IL-3 ⫹ CD40L, respectively. Then the CD11c⫹ and CD11c⫺ DCs were cultured with
CD45RA⫹ naive allogeneic CD4⫹ T cells isolated from either
adult blood or cord blood. After 5 days of culture, the cells were
restimulated with PHA overnight, and the secondary supernatants
were harvested and assayed for cytokines. As shown in Fig. 5, both
CD11c⫹ and CD11c⫺ DCs could induce the production of IFN-␥,
IL-4, and IL-10. However, the CD11c⫺ DCs consistently elicited
greater levels of IL-10 than the CD11c⫹ DCs (Fig. 7b). In addition, the levels of IFN-␥ induced by the CD11c⫹ DCs were significantly higher than those induced by the CD11c⫺ DCs. Thus,
the CD11c⫹ and CD11c⫺ DC subsets appear to elicit distinct Th
cytokine profiles, although the CD11c⫹ DCs do not induce a
strong Th1 response, as observed with monocyte-derived DCs (5).
It is conceivable that the induction of IL-10 production by the
CD11c⫺ DCs exerts a regulatory effect on T cell proliferation,
ultimately resulting in T cell anergy (30). In G-CSF-treated donors, where this CD11c⫺ subset is preferentially expanded, this
may result in a dampening of T cell responses. This is consistent
with the well-known anti-inflammatory effects of G-CSF in suppressing T cell proliferation in vitro (17–22). This is also consistent with data from murine allogeneic transplantation models,
which suggest that G-CSF treatment may also have direct effects
on donor T cell function by polarizing toward a Th2-cytokine
phenotype (17).
In summary, our data demonstrate that FL and G-CSF are potent
mobilization factors of distinct DC subsets in vivo. FL mobilizes
both the CD11c⫹ and the CD11c⫺ DC subsets, whereas G-CSF
preferentially mobilizes the latter. The effects of G-CSF on mobilizing the CD11c⫺ subset are consistent with a recent report (32).
These two DC subsets elicit distinct profiles of cytokines in T cells.
In mice, cytokines such as FL and GM-CSF can act as potent
adjuvants that differentially skew the Th1/Th2 balance in vivo (3).
It is now important to consider whether such cytokines can also
enhance T and B cell responses in humans. In particular, it is of
great importance to investigate whether FL and G-CSF can elicit
distinct types of immune responses in healthy humans.