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Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1598-1607
Proliferation in Monocyte-Derived Dendritic Cell Cultures Is
Caused by Progenitor Cells Capable of Myeloid Differentiation
By
Lois L. Cavanagh,
Russell J. Saal,
Karen L. Grimmett, and
Ranjeny Thomas
From the Centre for Immunology and Cancer Research, the Department of
Medicine, University of Queensland; and the Department of Haematology,
Princess Alexandra Hospital, Queensland, Australia.
 |
ABSTRACT |
Dendritic cells (DC) can be generated by culture of adherent
peripheral blood (PB) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). There is
controversy as to whether these DC arise from proliferating precursors
or simply from differentiation of monocytes. DC were generated from
myeloid-enriched PB non-T cells or sorted monocytes. DC generated from
either population functioned as potent antigen-presenting cells. Uptake
of [3H]-thymidine was observed in DC cultured from
myeloid-enriched non-T cells. Addition of lipopolysaccharide or tumor
necrosis factor- led to maturation of the DC, but did not inhibit
proliferation. Ki67+ cells were observed in cytospins of
these DC, and by double staining were
CD3 CD19CD11cCD40
and myeloperoxidase+, suggesting that they were myeloid
progenitor cells. Analysis of the starting population by flow cytometry
demonstrated small numbers of
CD34+CD33CD14 progenitor
cells, and numerous granulocyte-macrophage colony-forming units were
generated in standard assays. Thus, production of DC in vitro from
adherent PB cells also enriches for progenitor cells that are capable
of proliferation after exposure to GM-CSF. Of clinical importance, the
yield of DC derived in the presence of GM-CSF and IL-4 cannot be
expanded beyond the number of starting monocytes.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
DENDRITIC CELLS (DC) are derived from
CD34+ progenitors in the bone marrow (BM).1
Human peripheral blood (PB) DC are closely related to monocytes. Their
common precursor can be identified by hematopoietic progenitor
colony-forming assay, and is derived from the common
granulocyte-macrophage colony-forming unit (CFU-GM).2,3 DC
circulate in the blood in very small numbers, and migrate into various
tissues as a part of steady-state traffic. After contact with antigen,
DC migrate to draining lymph nodes, where they undergo functional
maturation to become specialized antigen-presenting cells (APC). DC are
capable of activating naive T cells in the initiation of immune
responses,4 and therefore are desirable APC for clinical
adoptive immunotherapy.
Investigations into the ontogeny of DC led to the observation that
purified CD34+ stem cells derived from either BM or blood
could be differentiated in vitro into functional APC by the addition of
the cytokines tumor necrosis factor- (TNF-) and
granulocyte-macrophage colony-stimulating factor (GM-CSF) to the
culture medium.2,3,5 The yield of DC generated from
CD34+ precursors could be augmented with c-kit
ligand,6,7 or more effectively by a complex cocktail of
cytokines, including c-kit ligand, transforming growth
factor- 1, and flt3-ligand.8,9 However, alternative
techniques have been developed for the generation of functional DC by
the culture of either adherent blood or BM cells in the presence of
GM-CSF and IL-4.10-12
Although the phenotype of DC derived from adherent mononuclear cell
culture was relatively immature, differentiation could be induced by
inclusion of TNF- or CD40-ligand, or monocyte-conditioned medium
late in the culture period.13-15 A number of studies
concluded that CD14+ monocytes gave rise to DC in the
presence of GM-CSF and interleukin-4 (IL-4).16-21 Despite
this consensus, there has been contention as to whether
monocyte-derived DC proliferate in the presence of GM-CSF and IL-4.
While it was initially suggested that these DC were derived from
proliferating precursors,10,13 subsequent investigations
failed to detect evidence of proliferation.21 The current
studies examined whether DC derived from monocytes had the potential to
proliferate under various conditions, and whether DC differentiation
was associated with a reduced proliferative capacity. These studies
show that proliferation in these cultures results from small numbers of
contaminating progenitor cells, and not proliferation of DC or their
monocyte precursors.
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MATERIALS AND METHODS |
Culture medium.
All cells were cultured in RPMI 1640 (GIBCO, Life Technologies,
Mulgrave, VIC, Australia) supplemented with 10% fetal calf serum (FCS;
CSL Ltd, Parkville, VIC, Australia), 0.3 mg/mL L-glutamine (Trace
Biosciences, Castle Hill, NSW, Australia), 0.12 mg/mL benzylpenicillin (CSL) and 10 µg/mL gentamicin (Delta West, Pharmacia and Upjohn, Spring Hill, QLD, Australia) at 37°C in 5% CO2-in-air.
Monoclonal antibodies (MoAbs).
The MoAbs used in this study are shown in
Table 1.
Cell isolation.
PB mononuclear cells (PBMC) were prepared as described.22
Briefly, mononuclear cells were isolated from either heparinized venous
blood from healthy volunteers or from buffy coats (Red Cross Blood
Transfusion Service, Brisbane, Australia) by density gradient
centrifugation over Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden)
at 170g for 45 minutes. PBMC were washed and incubated with
neuraminidase-treated sheep erythrocytes. The rosetting fraction
(ER+, T cells) was separated from the nonrosetting fraction
(ER, non-T cells) by density gradient centrifugation
on Ficoll-Paque; remaining red blood cells (RBCs) were removed by 1 mol/L ammonium chloride lysis, and cells were washed. Myeloid-enriched
non-T cells were produced by depletion of T, B, and natural killer (NK) cells from non-T cells, by incubation with MoAb against CD19, CD16, and
CD3 (OKT3), followed by goat anti-mouse Ig magnetic beads (Miltenyi
Biotech, San Francisco, CA), passage through a separating column in a
strong magnetic field (MACS; Miltenyi Biotech), and collection of the
unbound fraction. Eighty-five percent to 95% of the negatively
selected fraction were dendritic cell precursors or monocytes that
expressed CD33.23
Selection of cells by cell sorting.
For some experiments, pure populations of monocytes, DC, and B cells
were isolated by cell sorting. For B cells, T and NK cell-depleted
ER cells were incubated with CD19-phycoerythrin
(PE) for 30 minutes on ice, then washed. For DC and
monocytes, T, B, and NK cell-depleted non-T cells were incubated with
CD14-fluorescein isothiocyanate (FITC) and CD33-PE. Cells were sorted
using an EPICS Elite ESP flow cytometer (Coulter Electronics, Hialeah,
FL) by gating on either CD19+ cells (B cells), or
CD33+CD14bright cells (monocytes) or
CD33+CD14dim cells (DC) as previously
described.24 In some experiments a gate was set to include
all cells. These mock-sorted cells were used as a control for the
sorting procedure.
Culture of PBMC and monocytes.
PBMC were cultured to produce monocyte-derived DC as
described.10 Briefly, 5 × 106 PBMC were
cultured in 1.5 mL medium per well of a 24-well plate (Costar Corp,
Cambridge, MA). After 2 hours the cells were gently agitated and the
nonadherent cells removed. In preliminary experiments, adherent PBMC
were cultured in the presence of 800 U/mL GM-CSF and various doses of
IL-4. Optimum APC function was observed if the cells were cultured in
the presence of either 400 or 500 U/mL IL-4 (data not shown). The
adherent fraction was therefore cultured in fresh medium containing 400 U/mL IL-4 (Sigma) and 800 U/mL GM-CSF (Schering-Plough Pty Ltd, Baulkam
Hills, NSW, Australia) for 2 to 7 days. Myeloid-enriched non-T cells or
sorted monocytes were cultured similarly in 1.5 mL medium containing
IL-4 and GM-CSF. Approximately 2 × 106 cells were
cultured per well in 24-well tissue culture plates for 2 to 14 days.
After culture, the wells were washed with several changes of
phosphate-buffered saline (PBS) to harvest the loosely adherent cells.
Preparation of T cells.
T cells were isolated by collecting ER+ cells that passed
through a nylon wool column, followed by magnetic immunodepletion of
cells expressing CD14, CD16, CD19, or HLA-DR as described above. Greater than 98% of the recovered cells expressed CD3 by flow cytometric analysis.
Mixed lymphocyte reaction (MLR).
Harvested APC were washed and resuspended at 5 × 106
cells/mL in medium containing 0.08 mg/mL mitomycin C (Sigma) to inhibit cell proliferation. Cells were incubated at 37°C in the dark for 20 minutes, then washed three times in Hanks' balanced salt solution (HBSS). Various numbers (102 to 104 per well)
of APC were incubated in triplicate in round-bottom 96-well tissue
culture plates (Costar) with 105 allogeneic T cells for 5 days at 37°C. DNA synthesis was measured by incorporation of
[3H]-thymidine ([3H]-TdR 1 µCi/well;
Amersham, Buckinghamshire, UK) added during the final 18 hours of the
culture period. Cells were harvested onto glass fiber filtermats by an
automated cell harvester (LKB Wallac, Turku, Finland), and
incorporation of [3H]-TdR was determined by liquid
scintillation spectroscopy. Results are expressed as the mean cpm ± SD.
In vitro differentiation of DC.
Cells cultured in the presence of GM-CSF and IL-4 were stimulated in
vitro by the addition of 100 U/mL TNF- (National Institute for
Biological Standards and Control, Hertfordshire, UK) or 1 µg/mL
lipopolysaccharide (LPS) (Sigma) for the final 24 hours of the culture
period.
Immunophenotyping of cells by flow cytometry.
Freshly isolated or cultured cells were stained with either FITC- or
biotinylated-MoAb or unconjugated primary MoAb (Table 1) on ice for 30 minutes, then washed. Incubation with unlabeled MoAb was followed with
biotinylated-rabbit anti-mouse IgG (DAKO), and finally
streptavidin-conjugated-FITC (SA-FITC; DAKO) for 30 minutes.
Biotinylated-MoAb were followed by SA-FITC as described. Cells were
analyzed using an EPICS Elite ESP flow cytometer (Coulter) and data
analyzed using Winlist software (Verity Software House, Topsham, ME).
CD34+ progenitor cell determination by flow
cytometry.
Enumeration of progenitor cells in freshly prepared or cultured
myeloid-enriched non-T cells was performed by flow cytometry according
to ISHAGE guidelines.25 Briefly, cells were labeled with
CD45-FITC and CD34-PE, and analyzed by sequential gating using side
scatter and fluorescence properties of at least 50,000 events.
Cell proliferation assay.
In some experiments, proliferation of APC cultured with cytokines was
determined by [3H]-TdR incorporation. Cultured cells were
washed three times with PBS to remove residual cytokine-containing
medium, then plated at varying cell numbers (104 to
105) per well in medium without cytokines. Control cells
were treated with mitomycin C before culture. All wells were pulsed
immediately with [3H]-TdR, cultured for 18 hours, then
harvested as described. Cell proliferation is expressed as mean cpm ± SEM for triplicate wells.
Detection of cyclin-dependent kinase-1 (cdk1) by
immunoblotting.
Cytoplasmic lysates were prepared from cells after varying times in
culture with the various cytokine combinations. Cells were resuspended
at 2 × 105 cells/5 µL of lysis buffer (0.1%
Triton-X-100 in Tris buffer), incubated on ice for 20 minutes with
occasional vortexing, then spun at 1,500 rpm for 10 minutes at 4°C.
The supernatant was collected and either stored at  20°C for
later use, or mixed with an equivalent volume of reduced Laemmli
loading buffer (Bio-Rad Laboratories Pty Ltd, Regents Park, NSW,
Australia) and boiled for 2 to 3 minutes. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed
using a Bio-Rad Protean II minigel apparatus.26 Proteins
were first run through a 5% acrylamide stacking gel, then separated in
a 12% acrylamide separating gel. Transfer of proteins from
SDS-polyacrylamide gels onto nitrocellulose membrane (Hybond-C;
Amersham) was performed using a Bio-Rad mini trans-blot apparatus. The
nitrocellulose filters (blots) were transferred to blocking solution
(5% skim milk powder in Tris buffered saline, TBS, containing 0.05%
tween 20). The blots were blocked either for 2 hours or overnight at
4°C, followed by incubation in anti-cdk1 or an
irrelevant control MoAb for 1 hour at room temperature. The blots were
washed three times in TBS-tween over 15 minutes, then incubated in
sheep anti-mouse Ig conjugated to horseradish peroxidase (HRP; Silenus
Laboratories, Hawthorn, VIC, Australia) for 1 hour. After washing, the
chemiluminescent ECL reaction (Amersham) was performed for 1 minute,
followed by exposure to X-OMAT AR scientific imaging film (Eastman
Kodak Co, Rochester, NY).
Immunohistochemistry.
105 freshly isolated or cultured cells were cytospun onto
Vectabond-coated slides (Vector Laboratories Inc, Burlingame, CA). Cytospins were air dried, then fixed in absolute acetone for 2 minutes
at room temperature, and stained or stored frozen until required. For
single stains, cytospins were incubated in blocking solution (5%
FCS/5% normal sheep serum) for 20 minutes, then incubated with primary
MoAb, or irrelevant control antibody (mouse IgG1 or rabbit Ig negative
controls; DAKO) for 60 minutes. Slides were washed in TBS between each
step. Endogenous peroxidase was blocked by incubation in 0.5%
H2O2. This was followed by incubation with either biotinylated rabbit anti-mouse Ig (DAKO) or biotinylated swine
anti-rabbit Ig (DAKO) for 30 minutes, then horseradish peroxidase (HRP)-streptavidin complex (DAKO) for 30 minutes. The HRP product was
developed with liquid DAB+ substrate-chromogen system
(DAKO). For double staining, the above method was followed, except that
after addition of the HRP complex, slides were again blocked, followed
by the second primary MoAb or irrelevant control antibody for 60 minutes, and then incubated in appropriate biotinylated secondary
antibody for 30 minutes. Slides were incubated with streptavidin
ABC-alkaline phosphatase complex (DAKO) for 30 minutes, then the
peroxidase substrate. Finally, Vector red substrate (Vector
Laboratories) was used to develop the alkaline phosphatase reaction.
Cytospins were counterstained with Mayer's hematoxylin (Sigma) to
demonstrate cell nuclei.
For myeloperoxidase staining, cytospins were prepared as described, air
dried, then fixed in 10% formal-ethanol solution. Slides were
incubated with filtered Kaplow's reagent for 30 seconds, washed in tap
water, and counterstained in 20% Giemsa. Slides were finally washed in
buffered distilled water.27
CFU-GM assay.
Freshly isolated myeloid-enriched non-T cells, or DC generated in
culture for 11 days, were plated on semi-solid methylcellulose medium
containing IL-3, stem cell factor, and GM-CSF (Methocult GF H4534; Stem
Cell Technologies Inc, Vancouver, BC, Canada) at a concentration of 3 × 105/plate, and incubated for 14 days. CFU-GM
colonies (>50 cells) were counted by visual examination of the
plates, according to the method described by the Terry Fox Laboratory
Media Preparation Service.28 The mean colony count per
105 cells was calculated.
| |
RESULTS |
CD14bright monocytes are responsible for the potent
allostimulatory function of PBMC cultured in GM-CSF and IL-4.
Adherent PBMC develop potent APC function after prolonged incubation in
the presence of GM-CSF and IL-4. It has previously been shown that
CD14+ monocytes sorted from either freshly isolated or
cultured PBMC give rise to DC in the presence of GM-CSF and
IL-4.18,20 To compare the functional outcome of DC
generated from sorted monocytes, myeloid-enriched non-T cells, or PBMC,
these cell populations were cultured for 4 days in the presence of
GM-CSF and IL-4, and their APC function assessed in the allogeneic MLR.
As shown in Fig 1A, all preparations of
cells gave rise to APC of comparable function. Furthermore, as shown in
Fig 1B, only CD14bright monocytes cultured in GM-CSF and
IL-4 exhibited APC function comparable to unfractionated or mock sorted
PBMC. Neither the CD14/dim fraction nor
CD19+ B cells were effective APC after prolonged incubation
in the presence of GM-CSF and IL-4. The data show that monocytes give rise to APC after incubation in GM-CSF and IL-4, and that monocytes do
not require the presence of other mononuclear cells to differentiate into functional APC in the presence of GM-CSF and IL-4. Because there
were no functional differences observed between DC derived from any of
these starting populations, myeloid-enriched non-T cells (B and
NK-depleted ER) were used for the remaining
experiments.
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| Fig 1.
Monocyte-derived DC have potent APC function. DC prepared
from adherent PBMC, myeloid-enriched non-T cells, or sorted monocytes
(A), or from various sorted PBMC subpopulations (B), were cultured with
800 U/mL GM-CSF and 400 U/mL IL-4 for 4 days. Graded numbers (A) or
104 (B) mitomycin-treated APC were incubated with
105 allogeneic T cells for 5 days. Results are expressed as
mean cpm of triplicate samples.
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The previous experiments indicated that the presence of lymphocytes in
the starting population did not influence the function of the DC
generated by culture of adherent PBMC in the presence of GM-CSF and
IL-4. To determine whether phenotypic differences occurred, DC were
generated in the presence of GM-CSF and IL-4 for 7 days from either
adherent PBMC or sorted monocytes. DC were stained with various markers
of differentiation and analyzed by flow cytometry. As shown in
Fig 2, CD11b was not expressed by either DC
population, and CD14 expression was low. CD3 expression was associated
with DC generated from PBMC, but not from monocytes, due to
contaminating T cells within the cultures. In the case of DC derived
from PBMC, these CD3+ T cells were noted to be clustered
around DC by confocal microscopy (data not shown). The major
histocompatability complex (MHC) class II molecules HLA-DR and HLA-DQ
were expressed by both DC populations. However, DC derived from PBMC
also contained MHC class II lymphocytes. Although
CD40 and CD1a were expressed by both DC populations, CD80 and the DC
maturation marker CMRF-44 were not. CD86 was expressed by a small
percentage of monocyte-derived DC, and a somewhat greater percentage of
DC derived from PBMC. The data show that DC derived from monocytes and
PBMC exhibit different degrees of purity, and that adherent
PBMC-derived DC express higher levels of CD86 than sorted
monocyte-derived DC.
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| Fig 2.
Phenotype of DC generated after culture of adherent PBMC
or sorted monocytes. Cells were cultured in the presence of GM-CSF and
IL-4 for 7 days before analysis by flow cytometry. In each case
staining with an irrelevant control antibody is represented by the box
to the left of the histogram. Adherent PBMC are represented by the
solid line, and monocytes by the dashed line. Dead cells were excluded
by gating on forward and side scatter properties, and the remaining
viable cells were analyzed. In the case of DC generated from adherent
PBMC, contaminating lymphocytes were included. Histograms represent
data acquired from 10,000 events. Data are representative of four
separate experiments.
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Evidence of proliferation within cultures of monocyte-derived DC.
The next experiments were performed to determine whether
monocyte-derived DC are generated by differentiation from
nonproliferating monocytes, or whether a process of proliferation with
subsequent differentiation is involved. Myeloid-enriched non-T cells
were cultured for 4 days with or without cytokines, then washed, and incubated in medium containing [3H]-TdR for 18 hours.
Control cells were treated with mitomycin C to inhibit cell
proliferation. As shown in Fig 3,
proliferation was detectable only in cultures treated with GM-CSF and
IL-4. The low levels of [3H]-TdR uptake suggest either
that the cell turnover was low, or that only a subpopulation of cells
was indeed proliferating.
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| Fig 3.
Proliferation of cultured myeloid-enriched cells.
Myeloid-enriched non-T cells were cultured in the presence or absence
of GM-CSF and IL-4, washed, and reincubated for 18 hours with
[3H]-TdR. Control cells were cultured with GM-CSF and
IL-4 that had been treated with mitomycin C before assay. Cell
proliferation is expressed as the mean [3H]-TdR
incorporation of triplicate wells ± SD.
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To determine whether DC progenitor cells stopped proliferating as they
differentiated, myeloid-enriched non-T cells were cultured for 6 days
in medium supplemented with GM-CSF in the presence or absence of IL-4.
In some wells either LPS (1 µg/mL) or TNF- (100 U/mL) was added
for the final 24 hours of culture. As shown in
Fig 4, cells cultured with GM-CSF in the
presence or absence of IL-4 incorporated [3H]-TdR, and
this capacity was not inhibited by either LPS or TNF-. Donor-to-donor variability was observed in these assays, as illustrated in the three representative experiments illustrated in Figs 3 and 4.
The [3H]-TdR incorporation was particularly high in some
cultures in the presence of GM-CSF alone. The data show low-level
proliferation of cells in cultures supplemented by GM-CSF and IL-4. Of
importance, LPS and TNF-, which induce DC differentiation, do not
inhibit proliferation of those cells. In data not shown,
[3H]-TdR uptake was demonstrated after 2, 4, 6, and 9 days in culture, and addition of LPS at early and late time points did
not alter the proliferative capacity of the cells.
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| Fig 4.
Effect of LPS and various cytokines on the proliferative
response in DC cultures. Myeloid-enriched non-T cells were cultured in
the presence of various cytokine combinations, then washed and
reincubated for 18 hours with [3H]-TdR. Where added, LPS
or TNF were included during the final 24 hours of culture. Control
cells were cultured with GM-CSF and IL-4, and treated with mitomycin C
before assay. Proliferation is represented as the mean
[3H]-TdR incorporation of triplicate wells. The data
presented are representative of three separate experiments. ( ),
Medium; ( ), GM-CSF; ( ), GM-CSF + IL-4; ( ), GM-CSF/IL-4 + LPS; ( ), GM-CSF/IL-4 + TNF; ( ), control.
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To confirm that the addition of LPS induced DC differentiation
regardless of the time of addition, myeloid-enriched non-T cells were
analyzed by flow cytometry after either 2 or 7 days in culture. The
phenotype of each cell population was compared with that of freshly
isolated myeloid-enriched non-T cells
(Table 2). Cells were cultured either in
medium, or in the presence of GM-CSF, GM-CSF and IL-4, or GM-CSF and
IL-4 with LPS added for the final 24 hours of incubation. Nearly all
freshly isolated myeloid-enriched non-T cells expressed high levels of
CD14, and the majority of cells expressed HLA-DR but not HLA-DQ, CD86,
or CD1a. In culture medium alone, or with additional GM-CSF, 45% to
65% of monocytes retained surface expression of CD14 at 7 days. CD14
expression was somewhat downregulated by the addition of IL-4, and LPS
had little additional effect. While CD1a, CD86 and MHC class II
molecules were expressed late in the cultures, LPS addition
particularly after 2 days upregulated their expression. The most marked
effect of LPS after 7 days was the upregulation of CD86 expression. The
data indicate that LPS enhances DC differentiation whether added early
or later to cultures containing GM-CSF and IL-4. Taken together with
the previous experiments, it is clear that in the presence of GM-CSF,
IL-4, and LPS, proliferation and DC differentiation occur
simultaneously.
To confirm that cells were capable of proliferation in cultures of
monocyte-derived DC, the next experiments examined the activity of
cyclin-dependent kinase 1 (cdk1) an intracellular protein
expressed by cycling cellsand the expression of Ki67. Expression of
cdk1 was determined by immunoblotting.
Detergent-solubilized cytoplasmic lysates from 105
myeloid-enriched non-T cells that were either freshly isolated or that
had been cultured for 4 days with additional cytokines were prepared.
Cdk1 was detected in all samples
(Fig 5). However, by comparison with
equivalent cell numbers of the cycling cell line, BJAB, the levels of
cdk1 protein in freshly isolated or cultured myeloid cells
were relatively low. These data show that an essential cyclin-dependent
kinase is expressed at low levels by freshly isolated and by cultured
myeloid-enriched non-T cells. Thus, although cdk1
expression is observed in cultured cells, stimulation of proliferation
by GM-CSF and IL-4 is unlikely to be at the level of cdk1
induction.
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| Fig 5.
Cdk1 is expressed by freshly isolated and
cultured myeloid-enriched non-T cells. Myeloid-enriched non-T cells
were cultured in the presence of various cytokines with or without LPS
for the final 24 hours. Lysates from 105 cells were
immunoblotted as described in Materials and Methods using
anti-cdk1 MoAb. The cycling BJAB cell line was used as a
positive control. Data presented are representative of four separate
experiments.
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The low levels of [3H]-TdR uptake and of cdk1
might indicate either a low cell turnover, or a small percentage of
cells with a high level of proliferation. To determine which cells were
proliferating, expression of Ki67, a nuclear antigen, that is absent in
Go, was examined by immunohistochemical staining of
cytospins. Figure 6A represents Ki67
staining of cells cultured in the presence of GM-CSF and IL-4 for
varying lengths of time. Very occasional nuclear Ki67+
cells (brown, 0.78%) were observed among freshly isolated cells. Although the frequency of Ki67+ cells increased during
culture, only a small percentage of cells was cycling at any time
sampled. Similar numbers of cycling cells were found in cultures that
contained LPS. Taken together with the previous data, these results
indicate that proliferation was independent of DC differentiation. In
accordance with the [3H]-TdR incorporation, culture with
GM-CSF was associated with an increased proportion of Ki67+
cells (data not shown). From these data, it is likely that few, if any,
DC or DC precursors were proliferating.
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| Fig 6.
(A) Expression of nuclear Ki67 in DC cultures over time.
Cytospins were prepared from DC cultures at various times, and stained
with either irrelevant control antibody (bottom left) or Ki67 using an
indirect immunoperoxidase method as described in Materials and Methods.
Ki67 stains the nucleus brown. The percentage of Ki67+
cells is indicated in each panel. Original magnification × 130. (B)
Expression of Ki67 and phenotypic markers in DC cultures. Cytospins
were prepared from DC after 7 to 10 days in culture with GM-CSF and
IL-4 (e), or GM-CSF/IL-4 and LPS (a through d, f). In each case Ki67
stains the nucleus brown (arrow) and the other MoAb stains red: (a)
CD3, (b) CD20, (c) CD40, (d) CD1a, (e) p55, (f) p55. Original
magnification × 130.
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To investigate the phenotype of the Ki67+ cells, a variety
of lineage and DC differentiation markers was examined in combination with Ki67. In each case, the Ki67 staining is represented in brown, and
the other marker in red (Fig 6B). Although occasional CD3+
T cells and CD20+ B cells were identified, no lymphocyte
colocalized Ki67. DC differentiation markers CD40, CD1a, CD11c, and p55
were expressed by large numbers of cells and their expression was
upregulated by the addition of LPS. However, Ki67 did not colocalize
with any of these markers. From these data it is evident that the
proliferating cells were neither residual T or B cells, nor DC.
The nuclear morphology of Ki67+ cells in the cytospins was
reminiscent of early myeloid progenitors. Moreover, cells with this appearance were myeloperoxidase+ (data not shown). Because
myeloperoxidase is an early marker of the myeloid lineage, the presence
of CD34+ stem cells in the myeloid-enriched cell fraction
was investigated by flow cytometry. Whereas CD34+ cells
constituted 0.13% to 0.15% of PBMC, 0.27% to 0.75% of the myeloid-enriched cells were CD34+, representing a twofold
to fivefold enrichment. By three-color flow cytometry the
myeloid-enriched population contained approximately 0.72% ± 0.18%
(range, 0.32% to 1.15%) CD34+ cells, of which none
expressed either CD33 or CD14. The data indicate that progenitor cells
are present in the myeloid-enriched non-T fraction, and that their
frequency varies between donors. CFU-GM assays were performed to
determine their myeloid colony-forming potential. Freshly isolated
myeloid-enriched non-T cells gave rise to many CFU-GM colonies, whereas
myeloid-enriched cells that had been cultured previously for 11 days in
the presence of GM-CSF and IL-4 did not produce colonies in this assay;
rather, only single viable cells were observed across the plates. To
confirm that CD34+ cells were proliferating in these
cultures, CD34+ cells were separated by immunomagnetic
beads from myeloid-enriched non-T cells. The CD34-enriched fraction,
the CD34-depleted, and the starting populations were each cultured in
the presence of GM-CSF and IL-4. After 6 days, unfractionated cells
contained 1.7%, CD34-depleted fraction contained 0.6%, and the
CD34-enriched cells contained 5.5% Ki67+ cells.
To establish that proliferating progenitor cells did not influence the
yield of monocyte-derived DC, either myeloid-enriched non-T cells, or
these cells pretreated with mitomycin C, were cultured with GM-CSF and
IL-4 for 7 days, then analyzed by flow cytometry, or in the allogeneic
MLR. As shown in Table 3, the yield of DC
was unaffected by mitomycin C treatment. The data indicate that the DC
yield in cultures of myeloid-enriched cells ranges from 50% to 70% of
the starting population, and is not dependent on proliferating
progenitors. Furthermore, the APC function of DC derived from the
mitomycin-pretreated cells was equivalent to that of mock-treated cells
(Fig 7).
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| Fig 7.
Effect of mitomycin C pretreatment on APC function of
monocyte-derived DC. DC prepared from myeloid-enriched non-T cells
either treated with mitomycin C (+ mitomycin), mock-treated ( mitomycin), or untreated (control) were cultured for 6 days in the
presence of GM-CSF and IL-4. APC were incubated with 105
allogeneic T cells for 5 days. Results are expressed as mean cpm
(±SEM) of triplicate samples.
|
|
| |
DISCUSSION |
In the presence of GM-CSF and IL-4, PB blood monocytes differentiate in
vitro into cells with the phenotype and function of DC.10,18-20 The initial identification of proliferating DC
precursors in adherent blood cultures supplemented with GM-CSF and IL-4
suggested the possibilities that PB monocytes may be induced to
proliferate under these conditions, and that large yields of DC might
be generated by expansion from such precursors.10
Therefore, the current studies were performed to determine the nature
of the proliferating cells in such cultures. Initially, the function
and phenotype of DC generated from adherent PBMC, myeloid-enriched
non-T cells and sorted monocytes were compared directly. Whereas DC
generated from either pure monocytes or adherent starting populations
were similar in function, phenotypic differences were detected in that the DC generated from adherent PBMC expressed higher levels of CD86
than DC derived from sorted monocytes. In view of the clusters of
CD3+ T cells noted around DC derived from PBMC, it is
likely that these T cells contributed to DC activation by CD40
ligation.13,29-31 In previous reports, monocyte-derived DC
have varied widely in the expression of CD86. At least one factor in
this variability is likely to relate to the presence or absence of T
cells in the starting population.
The studies described here show that
CD34+CD33 progenitor cells, capable of
giving rise to CFU-GM under appropriate conditions, were responsible
for the low levels of proliferation in cultures of monocyte-derived DC.
Ki67 expression and [3H]-TdR incorporation was evident
over several days of incubation. Although there was some variability
between donors, the addition of GM-CSF was generally required for the
detection of a proliferative response. There was no enhancement of this
response with IL-4. Rather, IL-4 had a greater effect on the phenotype
of the monocytes in culture and the development of DC, as has been
previously described.16,32-34 In contrast, DC generated
from CD34+ precursors from BM or blood require the addition
of GM-CSF and TNF-. TNF-, in particular, is likely to be required
for the initial phase of DC development.35-37 Subsequently,
exclusion of TNF- and addition of IL-4 leads to optimal development
of DC.33,34
The inability of 11-day-cultured cells to generate CFU-GM, despite
ongoing proliferation, indicates that myeloid progenitors differentiated in vitro. This notion is supported by the increasing numbers of cells expressing myeloperoxidase during culture. In addition, IL-4 has been shown to strongly inhibit the development of
CFU-GM, but only weakly inhibits granulocyte development.38 It is unlikely that these proliferating precursors would develop into
DC in the absence of other cytokines, such as TNF-, which must be
included in CD34+ cell cultures for prolonged periods to
generate DC.2,3,5 The proliferative response observed in
cultures of myeloid-enriched cells is consistent with previous reports
of proliferating cells in cultures of adherent PBMC.10 In
contrast, other studies that used pure, or semi-purified monocyte
populations, found no evidence of proliferation.14,19,21
These data are all consistent with the idea that myeloid progenitors
proliferate in response to GM-CSF in these cultures. In the current
studies, the yield of DC generated from either monocytes or
myeloid-enriched non-T cells was not altered by blockade of mitosis by
mitomycin C. These data indicate that nonproliferating precursors give
rise to DC in these cultures. This has important implications for the
generation of monocyte-derived DC using GM-CSF and IL-4 alone, for
clinical trials. First, the maximum theoretical yield of DC can be
predicted from the monocyte count of each individual donor. Second, in
the absence of other cytokines that might induce differentiation of the
myeloid progenitor cells, the DC generated cannot be expanded beyond
the starting monocyte input.
Based on these results, it is interesting to consider whether DC
arising from proliferating cultures of murine blood and BM are indeed
derived from stem cells39,40 or whether they differentiate from a relatively more mature cell, particularly in the absence of
growth factors other than GM-CSF. In contrast to these mixed cultures,
the development of DC from proliferating precursors has been more
readily shown in cultures of purified human CD34+ cells or
murine thymic precursors.7,35,37,41-43 In general, however,
these cultures require more stringent growth factor supplementation. Recently, human CD34+ progenitor cells derived from cord
blood or BM have been shown to proliferate up to 25-fold in cultures
containing GM-CSF and TNF-.37,44,45 Furthermore, using a
two-step culture technique, DC precursors were observed to proliferate
at early timepoints. However, two distinct populations of DC precursors
were evident after 5 days in culture, and neither showed any
proliferative capacity.35 These data suggest a biphasic
differentiation of DC from progenitors whereby cells proliferate early,
but lose this capacity after lineage committment. Of interest, one of
the DC precursor populations could be identified by cell-surface
expression of CD14. From this precursor it was possible to generate
potent APC in the presence of GM-CSF and TNF-, and macrophage-like
cells in the presence of M-CSF.36 As has been shown for
circulating monocytes, these CD14+ cells were unable to
proliferate in response to a range of cytokines including GM-CSF and
M-CSF. However, in contrast to monocytes, this macrophage-like cell was
not dependent on IL-4 for APC function.21,35,46
Taken together, the previous and current studies indicate that the
generation of DC from PB monocytes by culture with GM-CSF and IL-4 does
not involve cell proliferation, but rather differentiation of DC.
Observed proliferation depends instead on CD34+ progenitor
cells with the potential for myeloid differentiation that are enriched
by various monocyte-enrichment procedures, such as adherence. Of
clinical relevance, the yield of DC obtained in such cultures can be
estimated from the starting number of monocytes evaluated in individual
donors.
| |
FOOTNOTES |
Submitted December 1, 1997;
accepted May 4, 1998.
Supported by grants from the Queensland Cancer Fund, the National
Health and Medical Research Council of Australia. R.T. is supported by
the Arthritis Foundation of Queensland.
Address reprint requests to Ranjeny Thomas, MD, University
of Queensland, Department of Medicine, Princess Alexandra Hospital, Woolloongabba QLD 4102, Australia.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Drs P. Lipsky, D. Hart, E Langhoff, and N. Saunders for the
gifts of MoAbs; Dr K. MacDonald and Prof I. Frazer for helpful
discussions; and A. Pettit and Dr G. Thomas for excellent technical
assistance.
| |
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Abstract
Introduction
Abstract