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IMMUNOBIOLOGY
Increased dendritic cell number and function following continuous in vivo
infusion of granulocyte macrophage–colony-stimulating factor and interleukin-4
Saroj K. Basak, Airi Harui, Marina Stolina, Sherven Sharma, Kohnosuke Mitani, Steven M. Dubinett, and Michael D. Roth
Dendritic cells (DCs) are rare antigenpresenting cells that play a central role
in stimulating immune responses. The
combination of recombinant granulocyte
macrophage–colony-stimulating factor
(rGM-CSF) and recombinant interleukin-4
(rIL-4) provides an important stimulus for
generating DCs from murine bone marrow
precursors in vitro. Using miniature osmotic
pumps, we now demonstrate that continuous infusion of these cytokines for 7 days
had a similar effect in vivo, increasing the
number and function of splenic DCs. Administration of rGM-CSF/rIL-4 (10 ␮g/d each)
increased the concentration of CD11ⴙ DCs
by 2.7-fold and the absolute number of
splenic DCs by an average of 5.7-fold. DC
number also increased in peripheral blood
and lymph nodes. The resultant DCs exhibited a different phenotype and function than
those in control mice or mice treated with
rGM-CSF alone. rGM-CSF/IL-4 increased
both the myeloid (CD11cⴙ/CD11bⴙ) and
the lymphoid (CD11cⴙ/CD8␣ⴙ) subpopulations, whereas rGM-CSF increased
only myeloid DCs. DCs were highly
concentrated in the T-cell areas of white
pulp after rGM-CSF/IL-4 administration,
whereas they were diffusely distributed
throughout white pulp, marginal zones,
and red pulp in mice treated with rGM-CSF
alone. rGM-CSF/rIL-4 also significantly increased the expression of major histocompatibility complex (MHC) class I and MHC
class II on CD11cⴙ cells and increased
their capacity to take up antigens by
macropinocytosis and receptor-mediated
endocytosis. Splenic DCs generated in
response to rGM-CSF/rIL-4 were functionally immature in terms of allostimulatory
activity, but this activity increased after
short-term in vitro culture. Systemic treatment with rGM-CSF/rIL-4 enhanced the
response to an adenoviral-based vaccine
and led to antigen-specific retardation in
the growth of established tumor. We conclude that systemic therapy with the combination of rGM-CSF/rIL-4 provides a new
approach for generating DCs in vivo.
(Blood. 2002;99:2869-2879)
© 2002 by The American Society of Hematology
Introduction
Dendritic cells (DCs) develop from bone marrow precursors and
are distributed in limited numbers throughout peripheral tissues
and lymphoid organs. Although they represent only a small
percentage of mononuclear leukocytes, they play a sentinel role in
initiating and regulating immune responses.1,2 The use of cytokines
(granulocyte macrophage–colony-stimulating factor [GM-CSF],
interleukin-3 [IL-3], IL-4, tumor necrosis factor-␣ [TNF-␣]) and
receptor ligands (flt3-ligand, CD40-ligand) to promote DC differentiation has become an important strategy for enhancing immune
responsiveness and stimulating antigen-specific immunity.3-9 Of
these approaches, treating DC precursors with the combination of
GM-CSF/IL-4 has been the most widely studied. With human cells,
neither GM-CSF nor IL-4 generates DCs when used alone in
vitro.10-12 Despite the expression of GM-CSF receptors on myeloid
precursors and the activity of this cytokine as a DC survival factor,
GM-CSF does not promote functional differentiation into DCs. In
contrast, the combination of GM-CSF with IL-4 produces a
coordinated down-regulation of myeloid features and an upregulation of major histocompatibility complex (MHC) expression,
costimulatory molecules, antigen processing, and T-cell stimulatory activity.7,11 These effects are similarly observed in vivo when
cancer patients are treated with a combination of systemic GMCSF and IL-4 but not with GM-CSF alone.13-15
In contrast to results obtained with human cells, murine studies
suggest a more independent role for GM-CSF as a DC growth and
differentiation factor.16-18 Inaba et al16 cultured bone marrow
progenitors in GM-CSF and identified budding clusters of cells
expressing dendritic morphology, MHC class I, MHC class II,
CD11c, and DEC-205. These cells exhibited potent allostimulatory
activity consistent with their identification as DCs. Similarly, the
systemic administration of GM-CSF to mice increases DC number
in vivo. Hanada et al19 implanted GM-CSF–secreting tumors into
mice and observed increased numbers of splenic DCs when
compared to controls. The direct administration of GM-CSF alone,
in the form of a polyethylene glycol–modified molecule, was also
recently shown to increase the number of splenic DCs, but only the
myeloid subpopulation expressing CD11c and CD11b.20
Despite these independent effects of GM-CSF, there is evidence
that IL-4 still plays an important synergistic role in generating
mouse DCs. Labeur et al8 compared bone marrow progenitors
cultured in GM-CSF alone to those cultured with the combination
of GM-CSF/IL-4. The presence of IL-4 increased the expression of
From the Division of Pulmonary and Critical Care, Department of Medicine, the
Department of Microbiology, Immunology and Molecular Genetics, and the
Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los
Angeles; and the West Los Angeles Veterans Affairs Medical Center, CA.
Review Research Funds from the Department of Veteran Affairs.
Reprints: Saroj K. Basak, Division of Pulmonary and Critical Care, Department
of Medicine, UCLA School of Medicine, Los Angeles, CA 90095-1690; e-mail:
[email protected].
Submitted February 22, 2001; accepted December 3, 2001.
Supported by grants from the Tobacco-Related Disease Research Program of
California (#7RT-0040), the Parker B. Francis Foundation (S.K.B.), the
Jonsson Comprehensive Cancer Center/UCLA, and the National Institutes of
Health/National Cancer Institute (CA 85686, IP50 CA90388) and by Merit
BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
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.
© 2002 by The American Society of Hematology
2869
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2870
BASAK et al
MHC, CD40, CD80, CD86, and DEC-205. Cells cultured with
GM-CSF/IL-4 produced 3-fold more IL-12 than those cultured in
GM-CSF, and they stimulated greater T-cell proliferation in
response to alloantigens or OVA peptide. Others have reported that
DCs grown in low concentrations of GM-CSF alone are tolerogenic, whereas those generated in response to GM-CSF/IL-4 are
not.20 Gunji et al22,23 failed to observe tumor rejection in mice
inoculated with tumor cells producing GM-CSF or IL-4 alone, but
they noted tumor rejection and the development of tumor-specific
immunity in mice injected with a combination of GM-CSF– and
IL-4–producing tumors.
To clarify the role of IL-4 on the differentiation and expansion
of mouse DCs in vivo, we used miniature osmotic pumps to deliver
continuous infusions of GM-CSF, either alone or in combination
with IL-4. Spleens and lymph nodes from cytokine-treated mice
were evaluated for evidence of DC differentiation and function.
The combination of GM-CSF/IL-4 increased the number of
myeloid (CD11c⫹/CD11b⫹) and lymphoid (CD11c⫹/CD8␣⫹) DCs,
whereas GM-CSF alone expanded only on the myeloid subset.
Immunohistology revealed a focal concentration of DCs within the
white pulp of splenic follicles in mice treated with GM-CSF/IL-4,
whereas GM-CSF by itself produced a more diffuse response. DCs
generated in vivo with rGM-CSF/rIL-4 expressed higher levels of
MHC class I and class II and a greater capacity for endocytosis,
macropinocytosis, and allostimulatory activity. Finally, in an
immunotherapy model, combining systemic rGM-CSF/rIL4 with
an adenoviral-based vaccine significantly slowed tumor growth in
an antigen-specific manner, whereas systemic administration of
rGM-CSF had no effect. Our results demonstrate important synergistic effects on DC differentiation and function when GM-CSF
and IL-4 are administered together in vivo.
Materials and methods
Mice
Male C57BL/6 and BALB/c mice, 8 to 12 weeks old (Charles River
Laboratories, Wilmington, MA) were housed in a pathogen-free vivarium
and fed ad libitum. Procedures involving mice were approved by the UCLA
and the West Los Angeles VA animal research committees.
Cytokines
Recombinant mouse GM-CSF (rGM-CSF; 2.4 ⫻ 108 U/mg) and IL-4
(rIL-4; 1 ⫻ 108 U/mg) were provided by the Schering-Plough Research
Institute (Kenilworth, NJ).
In vivo generation of DCs
C57BL/6 mice were treated with a 7-day continuous infusion of rGM-CSF,
alone or in combination with rIL-4, administered by mini-osmotic pump
(model 1007D; Alza, Palo Alto, CA). In brief, osmotic pumps were loaded
under sterile conditions with rGM-CSF (2-20 ␮g/mL), rIL-4 (2-20 ␮g/mL),
or both and were implanted in subcutaneous tissue over the mid-back.
Control animals received pumps loaded with diluent alone (0.9% saline).
The presence of circulating rGM-CSF and rIL-4 was determined on day 7
serum samples by specific enzyme-linked immunosorbent assay, as described by the manufacturer (BioSource International, Camarillo, CA).
Spleen cell preparation and phenotyping
Single-cell suspensions from control and experimental spleens and lymph
nodes were prepared by cutting the organs into small pieces and then
digesting them with 1 mg/mL type II collagenase (Worthington Biochemical, Freehold, NJ) and 0.02 mg/mL bovine pancreatic DNAse (Boerhinger
BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
Mannheim, Mannheim, Germany) for 30 minutes at room temperature.
Digestion mixtures were then treated with 0.1 M EDTA (Sigma, St Louis,
MO) for 5 minutes and were centrifuged to remove tissue debris, and cells
were washed in RPMI 1640 (Irvine Scientific, Santa Ana, CA) containing
2% fetal calf serum (FCS; Omega Scientific, Tarazana, CA). Red blood
cells were depleted by hypotonic shock. For fluorescence-activated cell
sorter (FACS) analysis, cell surface Fc receptors (FcR) were first blocked
by incubation with anti-FcRII␥ monoclonal antibody (mAb) (clone 2.4G2;
ATCC, Rockville, MD) for 30 minutes at 4°C. T cells, B cells, and NK cells
were identified by incubation with biotinylated Thy1.2, B220, or NK1.1
mAb (Caltag Laboratories, Burlingame, CA) for 30 minutes at 4°C and
labeling with fluorescein isothiocyanate (FITC)–conjugated streptavidin
(Caltag Laboratories). DC subsets were identified by incubation with FITC,
allophycocyanin (APC)–, or phycoerythrin (PE)–conjugated mAb directed
against CD11c, CD11b, CD8␣, MHC class I, or MHC class II (PharMingen,
San Diego, CA). After washing in phosphate-buffered saline (PBS) with
2% FCS, cells were fixed in 1% paraformaldehyde. The acquisition of
103 to 105 events was performed on a FACStar cytometer (Becton
Dickinson, San Jose, CA), and results were analyzed using Cellquest
software (Becton Dickinson).
In some experiments, DCs were further enriched by the depletion of T
and B cells. Single-cell suspensions of spleen cells were depleted of RBCs
and incubated with purified mAbs against Thy-1.2 and B220 for 30 minutes
at 4°C (PharMingen). Cells were then incubated for 30 minutes at 37°C in
serum-free RPMI 1640 medium containing 10% rabbit complement
(Sigma, St Louis, MO). After washing, the remaining cells were used as an
enriched population of DCs.
Histology and immunohistology
Spleens from control, rGM-CSF, and rGM-CSF/rIL-4–treated mice were
embedded in Tissue-Tek OCT compound (Miles, Elkhart, IN) by freezing in
liquid N2 and stored at ⫺80°C. Six-micrometer sections were cut on a
cryostat (Reichert Jung, Cambridge Instruments GmbH, Germany) and
were mounted on poly-L-lysine–coated slides. Sections were air dried
overnight, fixed in 10% formalin (Sigma) for 10 minutes, and stained with
hematoxylin (Fisher Scientific) and eosin (Sigma) or for immunohistology
as described for each antibody combination.
Dual detection of CD11c and CD11b was performed by washing slides
with PBS and blocking endogenous peroxidase activity with 0.3% H2O2 (10
minutes). Sections were blocked for 20 minutes with PBS containing 5%
bovine serum albumin (Fisher Scientific, Springfield, NJ) and 1% goat
serum (Jackson Immuno Research, West Grove, PA), rinsed, and stained
with anti-CD11c (PharMingen) for 1 hour. After washing, biotinylated goat
antihamster antibody (PharMingen) was added for 30 minutes. Specific
antibody binding was visualized by treating with a peroxidase substrate for
30 minutes using the Vectastain Elite ABC Kit (Vector Laboratories,
Burlingame, CA). Sections were again treated with H2O2 and were blocked
with 1% goat serum before incubation with rat anti-mouse CD11b
(PharMingen) for 1 hour. Sections were stained with biotinylated donkey
anti-rat antibody (Jackson Immuno Research) and were visualized with an
alkaline-phosphatase, fast-blue substrate using the Vectastain ABC-AP kit
(Vector Laboratories).
Dual detection of DEC-205 (rat antimouse DEC-205; Serotech, Raleigh, NC) and immunoglobulin Ig␤ (hamster antimouse Ig␤; PharMingen)
was performed in an analogous manner using species-appropriate blocking
serum and secondary antibody.
Measurement of endocytosis and pinocytosis
Enriched DC populations (1 ⫻ 106) prepared from spleens of control and
cytokine-treated mice were incubated with 1 mg/mL FITC-dextran (70 kd)
or Lucifer yellow (both from Molecular Probes, Eugene, OR) for 1 hour at
37°C. Control cells were treated in the same manner but were maintained on
ice to block energy-dependent uptake. Antigen uptake was terminated by
the addition of ice-cold PBS containing 0.1% azide. Cells were washed 3
times in PBS/1% FCS/0.1% azide, and extracellular antigen was removed
by incubation with a 1% trypsin–PBS solution (Sigma) for 3 minutes at
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BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
GM-CSF AND IL-4 GENERATE DCs IN VIVO
2871
37°C. Cells were counterstained with PE–anti-CD11c and APC–antiCD11b, and intracellular uptake of FITC-dextran and Lucifer yellow was
determined by flow cytometry.
or unpaired Student t test, as applicable to the assay conditions, with P
values as indicated.
Mixed-lymphocyte reaction
Results
Splenocytes from control and cytokine-treated C57BL/6 mice were depleted of B and T cells, as described above, to prepare DC-enriched
populations. Either they were directly tested as stimulators in mixedlymphocyte reaction (MLR) assays (fresh) or they were cultured for 36
hours in the presence of 20 ng/mL of rGM-CSF and rIL-4 before testing
(cultured). Allogenic T cells were prepared from the lymph nodes of 8- to
12-week-old BALB/c mice. In brief, single-cell suspensions from inguinal
and axillary lymph nodes were incubated with mAbs to B220, NK1.1, and
Gr-1 for 45 minutes at 4°C. Cells were then incubated at 37°C for 30
minutes in serum-free RPMI 1640 medium containing 10% rabbit complement (Sigma). T cells prepared by this method were at least 90% pure, as
determined by FACS analysis. MLR assays were performed in 96-well,
round-bottomed culture plates. Allogenic T cells (1 ⫻ 105 BALB/c) were
incubated with varying numbers of irradiated DC-enriched spleen cells (20
Gy) from control or cytokine-treated mice. Cells were cultured in 0.2 mL
RPMI 1640 containing 10% FCS and 10⫺4 M 2-ME (Sigma) in a
humidified CO2 incubator for 3 days. Culture wells were pulsed with 1.25
␮Ci (46.2 kBq) [3H] thymidine for 12 hours, and the cells were harvested
onto glass fiber sheets using an automated harvester. Proliferation was
determined by counting each sample in a liquid scintillation ␤-counter.
Background counts for T cells or DCs alone were always less than 200 cpm.
Tumor cell line
Systemic rGM-CSF and combined rGM-CSF/IL-4 increased
spleen and lymph node sizes and the concentration
of CD11cⴙ DCs
Continuous infusions of rGM-CSF and/or rIL-4 were delivered to
mice through miniature osmotic pumps, and dosing was initially
evaluated by changes in spleen size and the presence of CD11c⫹
DCs. Cytokine effects reached a plateau by day 7, and serum levels
reached 158.3/0 pg/mL (GM-CSF/IL-4) for rGM-CSF–treated
mice and 52.8/410 pg/mL (GM-CSF/IL-4) for rGM-CSF/rIL-4–
treated mice at the 10 ␮g/d dosing (data represent pooled serum
from 5 mice). When used alone, rGM-CSF increased spleen
cellularity and DC percentage in a dose-dependent manner up to 10
␮g/d. In contrast, rIL-4 produced minimal effects when infused by
itself but induced specific changes in spleen size and composition
when given in combination with rGM-CSF (Figure 1). At 10 ␮g/d,
rGM-CSF increased cellularity by 4-fold and the concentration of
CD11c⫹ DC by 2.1-fold compared with control mice (P ⱕ .05). In
contrast, the combination of rGM-CSF/rIL-4 increased spleen cell
number by only 2.1-fold, yet it enriched the percentage of CD11c
on average by 2.7-fold to as much as 25% of total spleen cells
The murine cell line E-22 (kindly provided by Dr S. Restifo, National
Cancer Institute, Bethesda, MD), a clone of the EL4 mouse thymoma line
stably expressing the LacZ gene, was used for in vivo tumor studies. Cells
were maintained in RPMI 1640, 10% heat-inactivated FCS, 0.03%
L-glutamine, 100 ␮g/mL streptomycin, 100 ␮g/mL penicillin, and 50
␮g/mL gentamicin sulfate (Life Technologies, Rockville, MD) in the
presence of 400 ␮g/mL G418 (Life Technologies).
Recombinant adenoviral vectors
Recombinant adenovirus Ad5.CMV-LacZ (AdV/␤-gal) was obtained from
Quantum Biotechnologies (Montreal, Quebec, Canada). Ad5.CMV-LacZ is
a first-generation, E-1–deleted adenovirus serotype 5 expressing the
bacterial LacZ gene under the control of the CMV-IE promoter/enhancer.
The control adenovirus, AdV/RR5 (kindly provided by Dr L. Butterfield,
University of California Los Angeles) is an E-1–deleted type 5 vector that
carries no reporter gene construct.24 Viral stocks were amplified on 293
cells. This was followed by CsCl purification, dialysis, and storage at
⫺80°C. The titer of viral stock was between 109 and 1013 plaque-forming
units (PFU)/mL by plaque assay on 293 cells.
In vivo adenoviral immunization and tumor
immunotherapy model
Eight- to 12-week-old C57BL/6 mice (n ⫽ 5/group) were injected subcutaneously with E-22 tumor cells (1 ⫻ 105 in 100 ␮L saline) in the right flank.
The following day, mice were implanted with osmotic pumps and were
treated with 7-day infusions of saline, rGM-CSF, or the combination of
rGM-CSF/rIL-4 (10 ␮g each per day) to increase the number of DCs in
vivo. Fourteen days after tumor inoculation, mice were immunized by
intraperitoneal injection with 1 ⫻ 108 PFU of either ADV/␤-gal, expressing
the LacZ transgene, or the control vector, AdV/RR5. Tumor volumes were
measured biweekly in mm3 (maximal length ⫻ width ⫻ height) with an
electronic caliper (Stoelting, Wheat Lake Wood Dale, IL).
Statistics
Data from individual representative experiments were presented as mean
values ⫾ SD, and data from multiple experiments were represented as mean
group values ⫾ SE. Differences between groups were determined by paired
Figure 1. Continuous infusion of rGM-CSF alone, or in combination with rIL-4,
increased spleen cellularity and the percentage of CD11cⴙ DCs. C57BL/6 mice
were treated for 7 days with rGM-CSF (GM, 10 ␮g/d) or the combination of rGM-CSF
and rIL-4 (GM/IL-4, 10 ␮g each per day) by subcutaneous osmotic pump. (A) Total
number of viable splenic leukocytes was determined by hemocytometer count on day
7. Treatment with GM alone produced an average 4.4-fold increase in cell number,
whereas combination rGM/rIL-4 produced an average 2.0-fold increase in cellularity.
Values represent mean ⫾ SE of 3 experiments. (B) Single-cell spleen suspensions
were stained with anti-CD11c-FITC mAbs, and the effects of cytokine treatment on
the percentage of CD11c⫹ DC were determined by FACS analysis. Three percent to
7% of control cells expressed CD11c, whereas 6% to 22% of cells from GM-treated
mice expressed CD11c and 8% to 25% of cells from rGM/rIL-4–treated mice
expressed CD11c. Values represent mean ⫾ SE of 3 experiments. *P ⱕ .05
compared with control; †P ⱕ .05 compared with GM; ‡P ⱕ .1 compared with control.
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2872
BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
BASAK et al
Table 1. Changes in spleen cell type and number after treatment with rGM-CSF and rGM-CSF/rIL-4
Total cells (⫻ 106)
Cells (%)
GM/IL-4
Control
GM
GM/IL-4
B220
47.6 ⫾ 14.8
Control
42.5 ⫾ 16
GM
32.4 ⫾ 5.6
25.1 ⫾ 12.6
55.9 ⫾ 22.6
22.1 ⫾ 1.9
Thy1.2
25.8 ⫾ 3.4
24.7 ⫾ 3.8
21.2 ⫾ 1.2
13.0 ⫾ 2.9
32.8 ⫾ 5.2
14.6 ⫾ 2.0
NK1.1
2.2 ⫾ 1.7
1.2 ⫾ 1.0
5.2 ⫾ 5.9
1.0 ⫾ 0.6
1.5 ⫾ 0.9
3.7 ⫾ 4.6
CD11b⫹
18.2 ⫾ 7.5
23.9 ⫾ 2.1
31.3 ⫾ 11.9
9.2 ⫾ 4.6
33.5 ⫾ 6.2
21.7 ⫾ 9.2
Total
93.8 ⫾ 8.5
92.4 ⫾ 18.4
90.1 ⫾ 14.9
48.2 ⫾ 14.2
123.7 ⫾ 25.3
62.2 ⫾ 13.6
Spleen cells were isolated and counted from mice treated with rGM-CSF or rGM-CSF/rIL-4 (10 ␮g/mouse per day for 7 days). Cells were analyzed by FACS for surface
expression of various markers of B cells (B220), T cells (Thy1.2), NK cells (NK1.1), and myeloid cells (CD11b). Data represent the mean ⫾ SD (n ⫽ 3 experiments).
(P ⱕ .05). Although rGM-CSF significantly increased the percentage of DCs, the response to combined rGM-CSF/rIL-4 was greater
in all experiments (P ⱕ .05).
A similar increase in total cell number was noted in the lymph
nodes of mice treated with rGM-CSF and rGM-CSF/rIL-4, with the
response always greater in mice receiving rGM-CSF/rIL-4 (1.7fold vs 2.7-fold increase compared to control, respectively; Tables
1 and 2). An increase in the percentage of CD11c cells in lymph
nodes also occurred, though the effects were smaller in magnitude
compared with those observed in spleen (1.3- to 2.3-fold increase
compared with control). The overall effect of cytokine treatment
was an increase in total cell number and CD11c content in spleen
and lymph nodes.
rGM-CSF increased all lineage-specific subsets, whereas
rGM-CSF/rIL-4 preferentially increased natural killer
cells and myeloid cells in spleen
Cytokine treatments increased spleen and lymph node cellularity, and
flow cytometry was used to determine changes in the B cell (B220⫹), T
cell (Thy1.2⫹), NK cell (NK1.1⫹), and myeloid (CD11b⫹) subsets
(Tables 1, 2). In spleen, treatment with rGM-CSF produced a proportional increase in all these subsets. In contrast, rGM-CSF/rIL-4 selectively increased the percentage and number of NK cells and myeloid
cells with little effect on the total number of B cells and T cells. These
differences suggest a more generalized proliferative response to rGMCSF and a more targeted effect of rGM-CSF/rIL-4 on spleen cell
populations. The response in lymph node was slightly different, with
both rGM-CSF and rGM-CSF/rIL-4 resulting in a proportional increase
in all cell subsets.
rGM-CSF/rIL-4 increased the number of myeloid
(CD11cⴙ/CD11bⴙ) and lymphoid (CD11cⴙ/CD8␣ⴙ) DCs
Murine CD11c⫹ DCs were divided into 2 primary subsets: lymphoid
DCs identified by the concurrent expression of CD8␣ and myeloid DCs
delineated by the expression of CD11b. Consistent with prior reports,17,20,21,25 treatment with 10 ␮g/d rGM-CSF for 7 days increased the
percentage of myeloid DCs by 3- to 5-fold, making them the predominant DC population in rGM-CSF–treated mice. The addition of rIL-4 at
10 ␮g/d increased this response further, boosting the concentration of
CD11c⫹/CD11b⫹ cells up to 7 times that found in control mice (Figure
2). The effects of cytokine treatment on the lymphoid subset in spleen
were different. rGM-CSF, when used as a single agent, did not increase
the percentage of CD11c⫹/CD8␣⫹ cells. However, when combined
with rIL-4 (both at 10 ␮g/d), the concentration of CD8␣⫹ DCs increased
by 2- to 3-fold over the concentration found in control mice (Figure 2).
These results were confirmed by 3-color analysis demonstrating that
CD11c⫹/CD8␣⫹ cells in rGM-CSF/rIL-4–treated mice also stained for
DEC-205, another lymphoid DC marker. The percentage of cells
expressing both DEC-205 and CD11c increased 2- to 3-fold in the
rGM-CSF/rIL-4 treatment group (Figure 2). The effects on DC composition were further magnified by the overall increase in spleen size. On
average, the number of myeloid DCs in the spleens of mice treated with
rGM-CSF increased approximately 8-fold compared with control mice,
averaging 7.1 ⫻ 106 CD11c⫹/CD11b⫹ cells per spleen (Figure 3). In
mice treated with rGM-CSF/rIL-4, the number of myeloid DCs
increased by approximately 5-fold, and the number of lymphoid DCs
increased by 3.7-fold. Similar, but not identical, trends were observed in
axillary and inguinal lymph nodes. Mice treated with rGM-CSF
exhibited a 2.2-fold increase in the number of myeloid DCs in lymph
nodes and a 3-fold increase in the number of lymphoid DC. In
rGM-CSF/rIL-4–treated mice, there was a similar 2-fold increase in
myeloid DCs in lymph nodes and a 4- to 6-fold increase in lymphoid
DCs (Figure 3). Dual-staining demonstrated that DEC-205 was primarily expressed on the CD8␣⫹ subset in spleen and lymph nodes,
consistent with its established expression on lymphoid DCs. In total, the
response to rGM-CSF was clearly biased toward increasing primarily
myeloid DCs, whereas the response to rGM-CSF/rIL-4 was more
balanced, significantly increasing myeloid and lymphoid subsets in
spleen and lymph node.
Localization of DCs by immunohistology
Spleens from control and cytokine-treated mice were stained with
hematoxylin and eosin or 2-color immunocytochemistry for evaluation of cytokine-related changes on DC number and distribution
(Figure 4). Anti-CD11b was used to identify cells of myeloid
Table 2. Changes in lymph node cell type and number after treatment with rGM-CSF and rGM-CSF/rIL-4
Total cells (⫻ 106)
Cells (%)
Control
GM
GM/IL-4
Control
GM
GM/IL-4
B220
14.3 ⫾ 1.9
20.8 ⫾ 1.9
16.0 ⫾ 3.2
1.0 ⫾ 0.7
1.8 ⫾ 0.6
2.2 ⫾ 0.8
Thy1.2
78.5 ⫾ 3.6
69.3 ⫾ 1.0
71.2 ⫾ 4.0
5.4 ⫾ 3.3
6.8 ⫾ 1.9
10.0 ⫾ 4.3
NK1.1
0.5 ⫾ 0.0
0.7 ⫾ 0.0
1.1 ⫾ 0.2
0.03 ⫾ 0.03
0.5 ⫾ 0.4
0.1 ⫾ 0.1
CD11b⫹
5.1 ⫾ 0.6
7.9 ⫾ 0.8
5.6 ⫾ 0.4
0.3 ⫾ 0.2
0.7 ⫾ 0.2
0.8 ⫾ 0.8
100.5 ⫾ 3.6
97.4 ⫾ 3.8
95.0 ⫾ 4.3
6.8 ⫾ 4.4
10.0 ⫾ 3.0
13.2 ⫾ 5.7
Total
Axillary and inguinal lymph nodes were recovered from mice and were processed in a manner identical to that used for spleen cells. Data represent the mean ⫾ SD (n ⫽ 3
experiments).
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BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
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onstrated hypertrophic PALS containing DEC-205⫹ cells and the
infiltration of DEC205⫹ cells into B-cell regions. The changes
observed in response to rGM-CSF alone were magnified by the
addition of rIL-4, resulting in intense staining and clustering of
DEC-205⫹ cells in PALS and within B-cell areas. As such,
immunohistology supported the findings obtained by FACS analysis and demonstrated distinct effects of rIL-4 on the distribution of
DC within T- and B-cell areas of the spleen.
Combined rGM-CSF/rIL-4 up-regulated MHC expression
and antigen uptake by splenic DCs
Figure 2. rGM-CSF and rGM-CSF/rIL-4 increased the percentage of myeloid
DCs (CD11cⴙ/CD11bⴙ) in spleen, but only rGMCSF/rIL-4 increased the percentage of lymphoid DCs (CD11cⴙ/CD8␣ⴙ). Mice were treated with rGM-CSF (GM) or
the combination of rGM-CSF/rIL-4 (GM/IL-4), as described for Figure 1. Spleen cells
were isolated on day 7, and FACS analysis was performed to determine the
percentage of total spleen cells expressing either a myeloid DC phenotype (upper
panel, stained with CD11b-PE and CD11c-FITC) or a lymphoid phenotype (middle
panel, stained with CD8␣-PerCP and CD11c-FITC). The percentage of cells expressing DEC-205, another lymphoid DC marker, were also determined (lower panel,
stained with CD11c-PE and DEC-205-FITC). The percentage of double-stained cells
for each marker set are identified. Results are from a single representative
experiment (n ⬎ 20).
origin, anti-DEC-205 to identify lymphoid DCs, anti-CD11c to
detect total DCs, and anti-Ig␤ to detect B cells.
Hematoxylin and eosin sections from control mice demonstrated well-organized follicular structures with normal distributions of red and white pulp. In cytokine-treated mice, these
structures were disrupted by a diffuse infiltration of mononuclear
leukocytes. High-power examination also revealed increased stromal tissue in cytokine-treated mice (not shown).
Dual staining for CD11b and CD11c (Figure 4, middle panel)
demonstrated several important features. First, it suggested the
presence of enlarged follicular structures in the spleens of cytokinetreated mice that were not discerned by simple hematoxylin and
eosin stain. This was confirmed by staining for Ig␤ (Figure 4, lower
panel). In control mice, CD11c⫹ DCs were present in small
numbers and were distributed in the peri-arteriolar lymphoid
sheaths (PALS) of the white pulp (W) and in the marginal zones. In
response to rGM-CSF, there was a striking increase in the CD11c⫹
population at these sites and extension of cells into the red pulp.
The pattern in mice treated with rGM-CSF/rIL4 was distinct. Cells
intensely stained for CD11c⫹ were concentrated within the center
of follicular structures, with only rare CD11c⫹ cells observed in the
marginal zones. In contrast, CD11b⫹ cells were predominantly
distributed within the marginal zones of all mice, regardless of
cytokine treatment, but were increased in number in response to
rGM-CSF. The DEC-205 marker was observed within the PALS
area of white pulp and, to a lesser degree, in the B-cell areas of
control spleen. Tissue sections from rGM-CSF–treated mice dem-
Expression of MHC class I and class II is essential for antigenpresenting activity, and it increases as DCs mature. Flow cytometry
was used to determine the mean florescence intensity (MFI) of
MHC class I and class II expression on lymphoid (CD11c⫹/
CD8␣⫹) and myeloid (CD11c⫹/CD11b⫹) DCs from control and
cytokine-treated mice (Figure 5). The most dramatic effects
occurred with MHC class I, which did not increase after treatment
with rGM-CSF alone but increased significantly after treatment
with rGM-CSF/rIL-4. These effects were most prominent on
lymphoid DCs, where MHC class I expression increased by 3- to
4-fold compared with myeloid DC, where expression increased by
only 30% to 75%. The response pattern was different with respect
to MHC class II, which increased moderately in both DC subsets in
response to rGM-CSF and increased further in response to rGMCSF/rIL-4. The up-regulation of MHC class I and MHC class II, in
response to systemic rGM-CSF/rIL-4, suggests cytokine-induced
maturation in vivo.
Figure 3. rGM-CSF increased the total number of myeloid DCs in mouse spleen,
whereas the combination of rGM-CSF/rIL-4 increased the number of myeloid
and lymphoid DCs in spleen and lymph nodes. Mice were treated with rGM-CSF
(GM) or the combination of rGM-CSF/rIL-4 (GM/IL-4), as described for Figure 1.
Spleen cells (A) and pooled axillary and inguinal lymph nodes cells (B) cells were
isolated, and FACS analysis was performed to determine the percentage of cells
expressing the myeloid DC (CD11c⫹/CD11b⫹) or the lymphoid DC (CD11c⫹/
CD118␣⫹) phenotype. Percentages were multiplied by the number of total spleen or
lymph node cells to determine the total spleen or lymph node cell number expressing
each phenotype. Data represent the mean ⫾ SE of 3 experiments. *P ⱕ .05
compared with control.
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BASAK et al
BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
Figure 4. Histology and immunohistology of DC subsets. Magnification, ⫻ 20. (top panel) Hematoxylin and eosin. Cytokine therapy resulted in a mixed cellular infiltrate and
distortion of normal follicle structure. (middle panel) CD11c (brown) and CD11b (blue) immunostain. Treatment with either GM alone or with GM/IL-4 resulted in enlarged and
somewhat disorganized follicles (arrows). GM increased the number of CD11b⫹ and CD11c⫹ cells distributed throughout the white pulp (W) and marginal zones. GM/IL-4
preferentially increased the CD11c⫹ population with a marked localization to the white pulp. (bottom panel) DEC-205 (brown) and Ig␤ (blue) immunostain. Ig␤ identifies B cells
and highlights the enlarged follicles in cytokine-treated mice. Control mice demonstrated light staining for DEC-205 within follicle centers. GM therapy was associated with an
increase in DEC-205 staining, consistent with the increase in follicle size. In contrast, rGM/rIL-4 resulted in intense staining of tightly packed DEC-205⫹ cells within the centers
of follicles.
Flow cytometry was also used to examine the effects of
cytokine therapy on endocytosis (uptake of FITC-dextran) and
macropinocytosis (uptake of Lucifer yellow). Similar to the effects
on MHC class I, the systemic administration of rGM-CSF did not
increase the pinocytotic activity of either myeloid (CD11c⫹/
CD11b⫹) or nonmyeloid (CD11c⫹/CD11b⫺) DCs, but rGM-CSF/
rIL-4 increased uptake by both subsets (Figure 6). Similar to the
effects of in vivo cytokines on the expression of MHC class II,
there was a modest but significant increase in endocytotic activity
by all DC subsets in response to rGM-CSF and another increase
after the addition of rIL-4. This up-regulation of endocytosis and
pinocytosis after systemic rGM-CSF/rIL-4 suggests cytokineinduced differentiation in vivo.
Allostimulatory capacity is primed by in vivo exposure
to rGM-CSF and rGM-CSF/rIL-4
Purified DCs from the spleens of control and cytokine-treated mice
were tested for their ability to stimulate allogenic T cells in a
one-way MLR (Figure 7). When tested fresh, without a period of in
vitro culture, only DCs from rGM-CSF–treated mice produced
significant T-cell proliferation above that of DCs from control
spleens. However, when DCs from the different groups were placed
in culture for 24 to 36 hours with 20 ng/mL rGM-CSF and rIL-4,
there was a dramatic increase in stimulatory activity from those
previously exposed to cytokines in vivo. T-cell proliferation
increased approximately 8-fold in response to in vivo rGM-CSF
and 10-fold in response to in vivo rGM-CSF/rIL-4, with the
response consistently higher in the rGM-CSF/rIL-4 group (P ⱕ .05).
Taken together, these results suggest that rGM-CSF and the
combination of rGM-CSF/rIL-4 increased the number of DCs and
primed them in vivo for enhanced T-cell stimulatory activity.
Administration of rGM-CSF/rIL4, but not rGM-CSF alone,
enhances the response to an adenoviral-based vaccine
and promotes antigen-specific tumor responses in vivo
To access the functional impact of cytokine therapy (rGM/CSF vs
rGM-CSF/rIL-4) on antigen-presentation and antitumor immunity
in vivo, an established tumor immunotherapy model was developed. E-22 tumor cells, expressing ␤-gal as a model tumor antigen,
developed into solid tumors in 100% of mice treated with
subcutaneous injection of 1 ⫻ 105 cells. Pre-immunization with a
single intraperitoneal administration of 109 PFU of AdV/␤-gal, but
not the control vector (AdV/RR5), imparted 100% protection from
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BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
Figure 5. rGM-CSF/rIL-4 increased the expression of MHC class I, whereas
rGM-CSF and rGM-CSF/IL-4 increased the expression of MHC class II on
splenic DCs. Mice were treated with rGM-CSF (GM) or the combination of
rGM-CSF/rIL-4 (GM/IL-4), as described for Figure 1. Spleen cells were stained with
fluorescent mAb to CD11c and CD8␣ (lymphoid DCs) or CD11c and CD11b (myeloid
DCs) and were examined for the expression of MHC class I (A) or MHC class II (B).
Only GM/IL-4 significantly increased the expression of MHC class I on lymphoid and
myeloid DC subsets. In contrast, both GM and GM/IL-4 significantly increased the
expression of MHC class II, with the highest expression on cells from mice treated
with GM/IL-4. Data from a representative experiment (n ⫽ 5). *P ⱕ .05 compared
with control; †P ⱕ .05 compared with GM. 䊐 indicates control; g, GM; ■, GM/IL-4.
subsequent tumor challenge and generated tumor-specific immunity (data not shown). However, in mice with established tumors,
vaccination with 109 PFU of AdV/␤-gal only slowed tumor
progression, and vaccination with 108 PFU had no detectable effect
(data not shown). To evaluate the role of cytokines in enhancing the
vaccine response, mice were injected with tumor and, 1 day later,
were treated with saline, rGM-CSF, or rGM-CSF/rIL-4 by miniosmotic pump. Subsequent vaccination with 108 PFU of AdV/␤-gal
significantly reduced the rate of tumor growth only in mice that had
been treated with rGM-CSF/IL-4 (Figure 8B). The response in
mice pretreated with rGM-CSF was identical to that in salinetreated controls (not shown), yielding no detectable effect on tumor
growth. The antigen-specific nature of the response was confirmed
in animals immunized with AdV/RR5 (Figure 8A). Vaccination
with this control vector, lacking ␤-gal, produced no effect on the
growth of E-22. In vitro cocultures of cytokines and tumor cells
were carried out to evaluate whether combined rGM-CSF/rIL-4
had a direct cytotoxic or cytostatic effect on the growth of E-22. No
effect on cell growth (as assessed by cell number and thymidine
uptake) was observed in the presence of cytokines in the range of
0.1 ng/mL to 1␮g/mL. These results suggest that pretreatment with
rGM-CSF/rIL-4 enhances the vaccine response and stimulates
antigen-specific, antitumor immunity in vivo.
Discussion
Various differentiation and maturation signals have been used to
generate DCs in vitro, including GM-CSF for stimulating murine
GM-CSF AND IL-4 GENERATE DCs IN VIVO
2875
progenitors from bone marrow or spleen,16-18 GM-CSF in combination
with IL-4 for stimulating human and murine precursors,7,8,10-12,26,27 or
these cytokines in combination with a variety of accessory stimuli,
such as tumor necrosis factor-␣ (TNF-␣), lipopolysaccharide
(LPS), CD40-ligand, or flt3-ligand.2-9 Of these approaches, the
combination of rGM-CSF/rIL-4 has emerged as one of the most
effective and frequently used. In vitro, rGM-CSF plays a primary
role in precursor survival, proliferation, and early differentiation,
whereas IL-4 promotes final differentiation along a DC pathway.28,29 IL-4 promotes the loss of macrophage features, increases
MHC expression, up-regulates costimulatory molecules, enhances
antigen uptake and processing, and synergizes with other factors to
increase IL-12 production.7,8,29,30 In the current study, we expanded
on these in vitro observations and demonstrated similar synergism
between rGM-CSF and rIL-4 when they were used to generate DCs
in mice in vivo.
An initial challenge in performing these studies was the short
half-life of rGM-CSF and rIL-4 when administered to mice in vivo.
In contrast to humans, in whom sustained cytokine levels persist
for up to 8 to 12 hours after a single subcutaneous injection,14,15,31
the half-lives for rGM-CSF and rIL-4 in the mouse are only 11
minutes20 and 5 minutes,32 respectively. These pharmacokinetics
likely explain why an earlier study, in which mice were treated with
10 ␮g/d GM-CSF and IL-4 as a single daily injection, failed to
elicit significant increases in DC.5 Others have approached this
problem by implanting gene-modified tumor cells secreting one or
both cytokines.19,22,23 These models, though delivering a complex
mixture of cytokines and other tumor-derived factors, confirmed
that in vivo exposure to GM-CSF alone, or in combination with
Figure 6. rGM-CSF and rGM-CSF/rIL-4 increased antigen capture by splenic
DCs. DC-enriched splenocytes from control and cytokine-treated mice were incubated with 1 mg/mL FITC-dextran or Lucifer yellow for 1 hour at 37°C, stained with
CD11c and CD11b to identify DC subsets, and analyzed by FACS for receptormediated endocytosis of FITC-dextran (A) or pinocytosis of Lucifer yellow (B), as
determined by mean fluorescence intensity (MFI). Uptake at 37°C is shown in the
upper bars, and uptake of cells incubated at 4°C is demonstrated by the lower bars.
Both DC subsets responded to in vivo GM and GM/IL-4 with significant increases in
endocytosis; the greatest response was observed in GM/IL-4–treated mice. In
contrast, only DCs from GM/IL-4–treated mice demonstrated increased pinocytosis.
Data are from a representative experiment (n ⫽ 5). *P ⱕ 0.05 compared with control;
†P ⱕ .05 compared with GM. 䊐 indicates control; g, GM; ■, GM/IL-4.
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BASAK et al
BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
GM-CSF for its effects on spleen, mouse studies suggest that
cytokine-induced changes in DC should be observed in the
circulation.20 In vitro studies also suggest that GM-CSF alone can
be used to generate murine DC,17,18,26 whereas human DCs require
additional differentiation factors such as IL-4.3,7,11,36
In contrast to rGM-CSF, the administration of rIL-4 as a single
agent did not increase spleen cellularity or DC number (data not
shown). This corresponds to in vitro studies in which IL-4 alone
failed to induce the proliferation or differentiation of myeloid
progenitors.7,37,38 When rIL-4 was administered in combination
with rGM-CSF, spleen cellularity increased significantly, but not to
the degree observed with rGM-CSF alone. This finding is consistent with prior studies in which IL-4 partially suppressed hematopoietic stem cell proliferation in response to GM-CSF.28,39 However, though limiting the expansion of T cells and B cells,
GM-CSF/IL-4 still allowed DCs to increase by approximately
6-fold compared with control spleens. Taken together, these facts
suggest a more targeted effect on DC proliferation and differentiation than occurs with GM-CSF alone.
In addition to increasing DC number, rGM-CSF/rIL-4 promoted
a more balanced expansion of myeloid and lymphoid DCs than
observed with rGM-CSF alone. Similar to other reports,20 we found
that myeloid DCs predominate de novo in the spleen at a ratio of
approximately 1.5:1 compared with lymphoid DCs and that this
Figure 7. Ability to stimulate allogeneic T-cell proliferation increased in spleen
cells from mice treated with either rGM-CSF or rGM-CSF/rIL-4. Spleen cells from
control and cytokine-treated C57BL/6 mice were enriched for DCs by depleting B
cells and T cells with antibody and complement. DC-enriched populations were
added directly to microwells containing 1 ⫻ 105 allogeneic T cells (A) or were cultured
for 36 hours in vitro with rGM-CSF and rIL-4 (20 ng/mL each) before testing in the
MLR assay (B). DCs from GM mice stimulated greater T-cell proliferation, and this
capacity increased with in vitro culture. In contrast, DCs from GM/IL-4 mice
demonstrated control levels of allostimulatory activity when directly added to the MLR
assay but dramatically higher activity after in vitro culture. Data are the mean ⫾ SE of
triplicate cultures and are representative of 5 separate experiments. *P ⱕ .05
compared with control. †P ⱕ .05 compared with GM.
IL-4, can increase the number of functional DCs. In fact, several
studies demonstrated greater induction of antitumor immunity
when both cytokines were administered together. Recently, Daro et
al20 successfully administered polyethylene-glycol modified GMCSF (pegGM-CSF) as a mechanism for generating DCs in vivo. To
extend this line of investigation and to study the combined effects
of GM-CSF/IL-4, we administered unmodified cytokines by miniature osmotic pumps. When implanted subcutaneously, these pumps
release cytokine(s) at a continuous rate for up to 1 to 2 weeks.33,34
This technique allowed us to examine the effects of GM-CSF and
IL-4 in the absence of other factors, such as tumor burden.
Using this approach, systemic rGM-CSF–produced marked
splenic hypertrophy with increases in most cell lineages including
DCs, monocytes, granulocytes, B cells, and T cells. Within the DC
population, rGM-CSF expanded only myeloid DCs, identified by
their co-expression of CD11c and CD11b and their lack of
expression of CD8␣.5,35 These DCs demonstrated modest increases
in MHC expression and endocytotic activity in comparison with
myeloid DCs from control mice, and they expressed significantly
more of these features than did lymphoid DCs. These findings are
essentially identical to those reported in mice treated with pegGMCSF or implanted with irradiated tumor cells secreting rGMCSF.19,20 However, it is interesting that these results differ from
those reported in cancer patients treated with rGM-CSF.13 In
humans, the systemic administration of rGM-CSF increases the
number of circulating monocytes but does not increase DC number
or promote circulating monocytes to acquire a DC phenotype or
function in vivo.13 Although these human studies did not examine
Figure 8. Systemic rGM-CSF/rIL-4 enhanced the response to an adenoviralbased vaccine and led to antigen-specific retardation in tumor growth. Mice
were inoculated subcutaneously in the flank with 1 ⫻ 105 E-22 mouse thymoma cells
that expressed the LacZ gene. Twenty-four hours later, osmotic pumps were
implanted to deliver 7-day administration of either saline, rGM-CSF, or the combination of rGM-CSF/rIL-4 (10 ␮g each cytokine per day). Fourteen days after tumor
inoculation, mice were immunized by intraperitoneal injection with 1 ⫻ 108 PFU of an
adenoviral vector expressing either the LacZ transgene (ADV/␤-gal) (B) or no
transgene (AdV/RR5) (A). Tumor sizes (mm3) were measured twice a week, and data
were presented as mean tumor size ⫾ SE for 5 mice per group. *P ⬍ .05 compared
with mice treated with either saline (not shown) or rGM-CSF. Tumor growth in mice
treated with saline (not shown) was identical to that in mice receiving rGM-CSF.
Representative experiment (n ⫽ 2).
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BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
ratio increased to roughly 7:1 in response to the effects of GM-CSF
on the myeloid subset. By comparison, exposure to rGM-CSF/
rIL-4 increased the number of myeloid DCs in spleen tissue by
approximately 6-fold and the number of lymphoid DCs by
approximately 3.7-fold, resulting in a more balanced myeloid-tolymphoid ratio. A similar pattern was observed in lymph nodes, in
which the combination of rGM-CSF/rIL-4 resulted in a greater
expansion of the lymphoid DC subset than occurred with rGMCSF alone. Although treatment with flt3-ligand also increases both
DC populations, it favors the expansion of lymphoid DCs in spleen
by approximately 2:1 over myeloid DCs.5,6,20,25
In untreated mice, myeloid and lymphoid DCs are present in
scant numbers and occupy distinct microenvironments within the
spleen.6,40,41 Lymphoid DCs primarily reside in T-cell areas of the
white pulp, whereas myeloid DCs are distributed throughout the
marginal zones with some extension into the red pulp. In response
to rGM-CSF in vivo, a striking splenic enlargement develops with a
diffuse increase in CD11c⫹ DCs throughout all regions, including a
significant increase in DEC-205⫹ cells infiltrating into the T-cell–
enriched PALS. These effects are similar to those described for
flt3-ligand6 and IL-1242 and suggest a general response pattern to
dendropoietic factors. However, in response to rGM-CSF/rIL-4,
DCs expressing CD11c and CD11b and those expressing DEC-205
became highly concentrated within enlarged PALS. This localization within T-cell zones and the distinct increase in staining
intensity observed in response to rGM-CSF/rIL-4 are similar to the
changes that occur in mice treated with lipopolysaccharide in
vivo.40 These changes are also reminiscent of the homing that
occurs when activated DCs migrate from the periphery to lymphoid
tissue.43 In contrast to other dendropoietic factors, these immunohistology results suggest that combined treatment with rGM-CSF/
rIL-4 produces the proliferation and mobilization of DCs and some
degree of activation and localization to T-cell–enriched areas.
Considerable controversy exists regarding the relative origins
and roles of myeloid versus lymphoid DCs. Although it has been
reported that CD11c⫹/CD8␣⫹ DCs closely resemble thymic DCs
and are derived from lymphoid-committed precursors,5,44-46 others
have reported that myeloid-committed precursors give rise to both
DC subsets in vivo and that myeloid DCs acquire the expression of
CD8␣ when trafficking from the periphery to regional lymph
nodes.43,47 As such, it is difficult to conclude anything from our
experiments about the types of progenitors that respond to GM-CSF/
IL-4 in vivo. Further studies are warranted to investigate this.
Similar controversy exists about the functional impact of the
up-regulation of myeloid versus lymphoid DCs. In the thymus,
CD8␣⫹ DCs play a role in clonal deletion, yet when CD11c/
CD8␣⫹ DCs are purified from flt3-ligand–treated mice, they
produce high quantities of IL-12 and stimulate strong Th1 responses.25,48 In contrast, myeloid DCs produce less IL-12 and
stimulate a mixed Th1–Th2 T-cell response.6,25 When directly
compared for their ability to stimulate antitumor immunity in vivo,
tumor cells secreting rGM-CSF were more potent than tumor cells
secreting recombinant flt3-ligand.49 This difference correlated with
the induction of exclusively myeloid DCs and a mixed Th1–Th2
response in GM-CSF–exposed animals versus a lymphoidpredominant DC response that stimulated exclusively Th1 cytokines in mice exposed to flt3-ligand. However, the co-administration of tumor cells secreting GM-CSF and IL-4 was found to
produce more potent antitumor responses than mice inoculated
with GM-CSF–secreting tumors alone.50 In addition, when a
combination of GM-CSF/IL-4 was administered to human subjects
GM-CSF AND IL-4 GENERATE DCs IN VIVO
2877
at 2-week intervals, antitumor responses were observed in patients
with metastatic prostate cancer.13
We also examined GM-CSF/IL-4 for evidence that it altered the
differentiation or activation state of resultant DC. Two clear effects
were observed. GM-CSF/IL-4 increased the expression of MHC
class I and MHC class II on myeloid and lymphoid DCs and
significantly increased their capacity for endocytosis and macropinocytosis. These changes closely recapitulate the effects when
GM-CSF and IL-4 are used together in vitro7,8,22, and suggest that
combined GM-CSF/IL-4 results in greater maturation along a DC
pathway. Despite these changes, freshly isolated DCs from both
treatment groups were still functionally immature when tested
immediately in an MLR assay. Their full T-cell stimulatory activity
was not observed until after a short-term in vitro culture. We
conclude from these experiments that though IL-4 promotes greater
differentiation along a DC pathway, it does not promote the
terminal activation or maturation required for them to function as
potent T-cell activators. Additional stimulatory activity might be
needed in the form of antigen or pathogen (virus, bacteria, parasite)
for the induction of a second signal to induce full maturation of
these DCs for vaccination strategy.
Finally, we examined the in vivo administration of rGM-CSF
and rGM-CSF/rIL-4 for their ability to enhance the response to a
vaccine and to stimulate antigen-specific antitumor immunity.
Neither rGM-CSF alone nor the combination of rGM-CSF/rIL-4
produced antitumor effects in a model of established tumor growth
(data not shown). We hypothesized that DCs generated in response
to these cytokines lacked access to the appropriate tumor antigens
required for initiating an antitumor response. To test this hypothesis, mice that had been inoculated with tumor were pretreated with
rGM-CSF or with combination rGM-CSF/rIL-4 and were vaccinated with an adenoviral vector expressing a model tumor-specific
transgene (␤-gal). In this setting, combined therapy with rGM-CSF/
rIL-4 followed by antigen-specific vaccination resulted in a significant and reproducible attenuation of tumor growth. The same
regimen, but using rGM-CSF alone or an empty adenoviral-vector
lacking the tumor antigen transgene (AdV/RR5), was ineffective at
generating any antitumor response. These results indicate that
rGM-CSF/rIL-4 is more efficient than rGM-CSF alone for stimulating an antitumor response and that the administration of a
tumor-specific antigen is required to direct the effects of rGM-CSF/
rIL-4 against the tumor. These results also lead us to speculate that
DCs generated in vivo by rGM-CSF/rIL-4, which express higher
levels of MHC and more avidly sample the environment for
antigens and which relocate to T-cell–enriched areas of spleen, are
more effective than their rGM-CSF–generated counterparts in
responding to an antigen challenge and stimulating T-cell responses. These findings agree with earlier studies in which a
mixture of tumor cell line(s) secreting rGM-CSF and rIL-4
generated more effective antitumor responses than did the same
number of tumor cells secreting only rGM-CSF.22,23 It was also
recently reported that tumors secreting rGM-CSF and rIL-4, but not
rGM-CSF alone, induce bone marrow mononuclear cells capable
of transferring immunity to syngenic mice.50 This response was
associated with a higher expression of higher DEC-205⫹ and
CD11c⫹ cells than observed after treatment with rGM-CSF–
secreting tumors, suggesting that a more balanced expansion of
lymphoid and myeloid DCs might play a role in this difference.
In summary, our studies demonstrate a synergism between rIL-4
and rGM-CSF, when administered in vivo, similar to that previously reported for these 2 cytokines when used to activate DC
precursors in vitro. In contrast to GM-CSF alone, which promoted
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2878
BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
BASAK et al
a broad-spectrum increase in spleen cells of all types, the combination of GM-CSF/IL-4 more selectively expanded the number of
DCs, including myeloid and lymphoid subsets. DCs generated in
response to GM-CSF/IL-4 expressed higher levels of MHC and
were capable of greater antigen uptake and T-cell stimulatory
activity. However, as experienced in vitro, additional activation is
required for these cells to express their full potency. Finally, we
demonstrate that the differences generated in response to treatment
with rGM-CSF/rIL-4, compared with rGM-CSF alone, enhanced
the animal’s response to an adenoviral-based vaccination and
induced antitumor immunity. Further studies are indicated to
determine the types of precursors that respond to rGM-CSF/rIL-4
and the impact of this therapy on immune responsiveness mediated
by different DC subpopulations in vivo. The striking similarity
between the effects we observed in mice and those previously
reported in humans13 suggest that the infusion of rGM-CSF/rIL-4
by miniature osmotic pumps may provide an important model for
developing and testing DC-based immunization strategies.
Acknowledgments
Flow cytometry was performed in the UCLA Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow Cytometry
Core Facility, which is supported by National Institute of Health
awards CA-16042 and AI-28697.
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2002 99: 2869-2879
doi:10.1182/blood.V99.8.2869
Increased dendritic cell number and function following continuous in vivo
infusion of granulocyte macrophage−colony-stimulating factor and
interleukin-4
Saroj K. Basak, Airi Harui, Marina Stolina, Sherven Sharma, Kohnosuke Mitani, Steven M. Dubinett and
Michael D. Roth
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