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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4652-4661
The Role of Tumor Necrosis Factor  in Modulating the Quantity of
Peripheral Blood-Derived, Cytokine-Driven Human Dendritic Cells and Its
Role in Enhancing the Quality of Dendritic Cell Function in
Presenting Soluble Antigens to CD4+ T Cells In Vitro
By
Bing-guan Chen,
Yijun Shi,
Jeffrey D. Smith,
David Choi,
James D. Geiger, and
James J. Mulé
From the Department of Surgery, University of Michigan Medical
Center, Ann Arbor, MI.
 |
ABSTRACT |
Because dendritic cells (DC) are critically involved in both
initiating primary and boosting secondary host immune responses, attention has focused on the use of DC in vaccine strategies to enhance
reactivity to tumor-associated antigens. We have reported previously
the induction of major histocompatibility complex class II-specific T-cell responses after stimulation with tumor
antigen-pulsed DC in vitro. The identification of in vitro conditions
that would generate large numbers of DC with more potent
antigen-presenting cell (APC) capacity would be an important step in
the further development of clinical cancer vaccine approaches in
humans. We have focused attention on identifying certain exogenous
cytokines added to DC cultures that would lead to augmented human DC
number and function. DC progenitors from peripheral blood mononuclear cells (PBMC) were enriched by adherence to plastic, and the adherent cells were then cultured in serum-free XVIVO-15 medium (SFM) for 7 days
with added granulocyte-macrophage colony-stimulating factor (GM-CSF)
and interleukin-4 (IL-4). At day 7, cultures contained cells that
displayed the typical phenotypic and morphologic characteristics of DC.
Importantly, we have found that the further addition of tumor necrosis
factor  (TNF) at day 7 resulted in a twofold higher yield of DC
compared with non-TNF-containing DC cultures at day 14. Moreover,
14-day cultured DC generated in the presence of TNF (when added at
day 7) demonstrated marked enhancement in their capacity to stimulate a
primary allogeneic mixed leukocyte reaction (8-fold increase in
stimulation index [SI]) as well as to present soluble tetanus toxoid
and candida albicans (10- to 100-fold increases in SI) to purified
CD4+ T cells. These defined conditions allowed for
significantly fewer DC and lower concentrations of soluble antigen to
be used for the pulsing of DC to efficiently trigger specific T-cell
proliferative responses in vitro. When compared with
non-TNF-supplemented cultures, these DC also displayed an increased
surface expression of CD83 as well as the costimulatory molecules, CD80
and CD86. Removal of TNF from the DC cultures after 2 or 4 days
reduced its enhancing effect on DC yield, phenotype, and function.
Thus, the continuous presence of TNF over a 7-day period was
necessary to achieve the maximum enhancing effect observed.
Collectively, our findings point out the importance of exogenous TNF
added to cultures of cytokine-driven human DC under serum-free
conditions, which resulted in an enhanced number and function of these
APC. On the basis of these results, we plan to initiate clinical
vaccine trials in patients that use tumor-pulsed DC generated under
these defined conditions.
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INTRODUCTION |
DENDRITIC CELLS (DC) are the most potent
antigen-presenting cells (APC) distributed in many tissues of the body
in humans and other species.1,2 DC can stimulate the
primary activation of T cells due to their enhanced capacity of
presenting immunogenic peptides in association with self-major
histocompatibility complex (MHC) class I1-4
and class II molecules,5-8 expression of coreceptor
molecules such as CD40, CD80, CD86,9 and
ICAM-3,10 as well as production of cytokines such as
interleukin-12 (IL-12).11,12 DC can also process both
exogenous protein13 and intracellular protein derived from
DNA transfection14 for presentation to T cells. Recently, it was reported that DC can directly modulate B-cell growth and differentiation via CD40 ligation15 and through the
production of soluble mediators such as IL-1, IL-6, and tumor necrosis
factor (TNF), which have been shown to be produced by DC or
DC-related cell lines.16,17
We and others have shown that DC pulsed with tumor-associated
antigen(s) in the form of whole cell lysates,5-7
peptides,1-4,18,19 proteins,20
RNA,21 or DNA14,22 could initiate primary MHC
class I- or class II-restricted T-cell responses that resulted in
antitumor effects in vitro and in vivo. On the basis of these studies,
attention has focused on the use of DC to enhance the host immune
response to tumor-associated antigens in clinical vaccine strategies in
humans with cancer.23,24 Thus, the identification of
approaches that would generate large numbers of DC with more potent
antigen-presenting capacity would be an important step in the further
refinement of vaccine approaches based on DC.
There is general agreement that DC can be generated from bone marrow-
and cord blood-derived CD34+ hematopoietic cell progenitors
with cytokines such as granulocyte-macrophage colony-stimulating factor
(GM-CSF), IL-4, and TNF.25,26 In addition, DC have been
derived from precursors in unfractionated27,28 and
CD34+ cell-depleted29 peripheral blood
mononuclear cells (PBMC) as well as from CD14+ blood
monocytes.17,30
Various recombinant cytokines have been used for the in vitro
generation of DC derived from several tissue sources (denoted cytokine-driven DC).1,2 In particular, the activity of
TNF in combination with GM-CSF with or without IL-4 has been
studied. For example, the addition of TNF to cultures has been shown
to inhibit spontaneous apoptosis of DC,31 to generate
CD83+ DC from CD14+ blood
monocytes,17 to cause phenotypic and functional maturation of DC,1,2,32,33 to increase DC mobility by rearrangement of
microfilaments and microtubules,34 and to augment the
capacity of DC to mediate delayed-type hypersensitivity
(DTH) responses in vivo.35 In these studies
and those of others,36,37 the level and specific type(s)
(positive or negative) of TNF effects on DC phenotype and function
have appeared to be dependent on the timing and duration of exposure of
the DC cultures to this particular exogenous cytokine.
We have recently initiated phase 1 clinical trials of autologous tumor
lysate-pulsed dendritic cells as a vaccine in adult and pediatric
patients with advanced solid tumors. In the current study, efforts were
focused on strategies to improve human DC function and overall
recovery. We have now investigated the effects of recombinant TNF on
the generation of human DC from PBMC under serum-free conditions in the
presence of GM-CSF and IL-4. Moreover, to increase the overall recovery
of DC, we have included DC precursors of both the
lymphoid38,39 and myeloid1,2 lineages known to
be present in PBMC by excluding the depletion of CD2+ cells
before culture. Collectively, our data show that TNF added for
extended time periods can mediate improved function and yield of human
cytokine-driven DC derived from PBMC.
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MATERIALS AND METHODS |
Media and reagents.
The medium used throughout the studies was XVIVO-15 (BioWhittaker,
Gaithersburg, MD). Tetanus toxoid (TT) was purchased as a sterile
liquid in vials from Connaught Laboratories, Ltd (North York, Ontario,
Canada). The activity of TT was 2250 Lf/12.2 mg/mL (lot no. TAS 319 R8), which was diluted further in XVIVO-15 to achieve 225 Lf/mL and
stored at 4°C before use. Candida albicans (CAD) was purchased from
Greer Laboratories, Inc (Lenoir, NC). Sterile, lyophilized vials of CAD
(lot no. XPLM73-7-X7-NV) were reconstituted to 100 µg/mL with
phosphate-buffered saline (PBS) and stored at  20°C before use.
Recombinant human IL-4 was kindly provided by Schering-Plough Research
Institute (Kenilworth, NJ). The specific activity was determined to be
6.35 × 107 IU/mg (lot no. 3-ENP-803). Vials were diluted
with XVIVO-15 to 50 µg/mL and stored at 80°C before use.
Recombinant human GM-CSF was kindly provided by Dr C. Reynolds (BRMP,
NCI, NIH, Frederick, MD). This GM-CSF (manufactured by Immunex Corp,
Seattle, WA) had a specific activity of 1.4 × 106 IU/250
µg. Lyophilized GM-CSF was reconstituted with sterile water, diluted
to 100 µg/mL with XVIVO-15, and stored at 80°C before use.
Recombinant human TNF was a gift from Dr D. Fraker (Department of
Surgery, University of Pennsylvania, Philadelphia, PA). This TNF
(manufactured by Knoll AG, Ludwigshafen, Germany) had a specific
activity of 8.2 × 106 U/mg protein as measured in the
L929 cytotoxicity assay without adding actinomycin D. Sterile,
lyophilized TNF (0.79 mg/vial) was first reconstituted with 1 mL
sterile water before further dilutions in culture medium.
Generation of DC from peripheral blood.
PBMC were obtained from leukapheresis specimens of normal donors after
Ficoll-hypaque density gradient separation. PBMC were washed twice in
Hank's balanced salt solution (HBSS; GIBCO BRL, Life Technologies,
Inc, Gaithersburg, MD) and were resuspended in XVIVO-15 medium at a
concentration of 1.6 × 106 cells/mL. Three milliliters of
this cell suspension (5 × 106 PBMC) were plated in
6-well tissue culture plates (Costar Corp, Cambridge, MA) and were
incubated at 37°C, 5% CO2 for 2 hours. The nonadherent
cells were gently removed by pipetting, and 3 mL/well of XVIVO-15
medium containing GM-CSF (100 ng/mL) and IL-4 (50 ng/mL) was added. All
cultures were maintained at 37°C, 5% CO2 for 7 days. At
day 7, culture medium was exchanged with fresh XVIVO-15 medium
containing GM-CSF/IL-4 with or without TNF (10 ng/mL). The cultures
were maintained for another 7 days. In some experiments, TNF was
removed 2 or 4 days later by harvesting the cultures and washing the
cells three times in medium. The washed cells were then replated in
fresh XVIVO-15 medium containing GM-CSF and IL-4 for an additional 5 or
3 days, respectively.
Enrichment of DC.
At day 14, the DC were harvested from culture and loaded onto
hypertonic 14.5% metrizamide columns and centrifuged for 10 minutes at
650g. The DC-enriched interface was collected and washed consecutively in 40 mmol/L and 25 mmol/L NaCl medium solution by
centrifugation. The interface contained an average 75% DC as defined
by typical morphology (veiled appearance) and surface phenotype by
fluorescence-activated cell sorting (FACS; coexpression of high level
CD86 and HLA-DR, but CD14 ). In some
experiments, DC were stained with fluorescein isothiocyanate (FITC)-conjugated HLA-DR and sorted by a Coulter EPICS-C Cell Sorter
instrument (Coulter Corp, Miami, FL) gated according to large cells (by
side and forward light scatter) that were positive for HLA-DR
expression.
Isolation of CD4+ T cells.
Human CD4+ T cells were purified from PBMC using the MACS
magnetic cell sorting system according to the manufacturer's
recommendations (Miltenyi Biotec, Sunnyvale, CA). Briefly, the PBMC
were resuspended in 80 µL MACS buffer for each 107 cells.
Anti-CD4 magnetic beads were added at 20 µL per 107
cells. The mixture was incubated at 4°C for 15 minutes, washed twice
with MACS buffer, and then placed on an MACS column in the magnetic
field; the CD4 cells were first eluted. The column was
removed from the magnetic field and the CD4+ cells were
collected. The purity of CD4+ cells was greater than 90%
by FACS phenotypic analysis.
Phagocytosis assay.
Two hundred thousand DC in 500 µL XVIVO-15 were incubated at 4°C or
37°C with 50 µL of a 10 mg/mL stock solution of different molecular
weight fluorescein-labeled dextran particles (Molecular Probes, Inc,
Eugene, OR). After 30 minutes to 3 hours of incubation, samples were
washed four times with PBS plus 2% fetal calf serum (FCS), fixed with
5% paraformaldehyde in PBS at 4°C, and examined for phagocytic
uptake with a FACScan (Becton Dickinson, Mountain View, CA).
Geographical mean fluorescence was calculated for all samples.
Allogeneic mixed leukocyte reaction assay.
One hundred thousand responding T cells from PBMC of allogeneic adult
donors were cultured in 96-well U-bottom microplates (Costar Corp,
Cambridge, MA) with different numbers of DC irradiated with 2,000 rad
of 137Cs generated gamma radiation (Gamma Cell 1000;
Nordion Corp, Kanata, Ontario, Canada). Cellular
proliferation was measured on day 5 by an 18-hour pulse with
[3H]thymidine at 1 µCi/well (3H-TdR,
6.7 Ci/mmol/L; DuPont-NEN, Boston, MA).
Antigen presentation assay.
To measure the efficiency of DC presentation of soluble antigen, 5 × 104 CD4+ T cells were cultured with 5 × 103 cultured DC (irradiated with 2,000 rad) in the presence
of different concentrations of TT or CAD in 200 µL XVIVO-15 medium in
96-well U-bottom microplates. Cultures were pulsed with 1 µCi/well
3H-TdR (6.7 Ci/mmol/L) on day 5. Cellular proliferation was
measured by 3H-TdR incorporation 18 hours later with a
liquid scintillation counter (Wallac 1205 Betaplate; Wallac,
Gaithersburg, MD). In some experiments, 1 × 105
CD4+ T cells were cultured with different numbers of
irradiated DC in the presence of a fixed concentration of TT (12.2 µg/mL) or CAD (10 µg/mL).
FACS analysis.
Cell surface staining used direct immunofluorescence (FACScan; Becton
Dickinson), and the samples were analyzed using Cell Quest software
(Becton Dickinson). Staining was performed with the following FITC- and
phycoerythrin (PE)-labeled monoclonal antibodies: PE-CD1a, FITC-CD3,
PE-CD11c, FITC-CD32, PE-CD33, FITC-CD80, FITC-CD86, FITC-mouse IgG1
(all from Pharmingen, San Diego, CA); FITC-CD14, FITC-HLA-DR, PE-mouse
IgG1 (all from Becton Dickinson); PE-CD83 (from Coulter/Immunotech,
Miami, FL); and FITC-mouse IgG2 and PE-CD4 (both from Sigma, St Louis,
MO). Primary antibodies were directed toward a panel of cell surface
markers and compared with the appropriate isotype-matched controls.
| |
RESULTS |
TNF expands the number of human DC and upregulates the
expression of costimulatory molecules.
To avoid the disadvantages of serum (eg, potential exposure to
pathogens and sensitization to irrelevant antigens), we have refined a
method to generate DC from human peripheral blood leukocytes (PBL) that
uses serum-free XVIVO-15 medium (SFM). Because DC have been shown to
develop from both myeloid and lymphoid
progenitors,17,27,28,30,38,39 we first attempted to
increase the yield of DC from PBMC by deleting the commonly used
E-rosetting step of DC purification to preserve precusors present in
the CD2+ cell fraction. DC precursors were enriched by
adherence to plastic and the adherent cells were then cultured for 14 days in SFM with added GM-CSF and IL-4. The DC generated in this
culture displayed phenotypic and morphologic characteristics of
mature DC. As shown in Fig 1, DC generated
under these culture conditions were either partially or entirely
positive for CD4, CD11c, CD32, CD33, CD86, and HLA-DR cell surface
markers but were negative for CD1a, CD3, CD14, and CD80 expression.
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| Fig 1.
Surface phenotype of metrizamide gradient-separated DC
detected by a panel of antihuman FITC- or PE-labeled antibodies and FACS analysis. DC were generated from PBMC cultured for 14 days in
XVIVO-15 serum-free medium containing GM-CSF and IL-4. The x-axis is a
logarithmic scale of fluorescence intensity and the y-axis represents
counts. DC showed high-level expression of MHC class II (HLA-DR), CD86,
and CD11c molecules.
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In a preliminary experiment, we found that 14-day DC treated with
TNF exhibited the characteristic cellular projections and veils,
which showed continuous extension, retraction, and reorientation. As
demonstrated by the seven representative experiments of Table 1, the further addition of TNF at day 7 resulted in a twofold higher yield of harvested DC compared with
non-TNF-containing DC cultures at day 14 (2.77 ×
105 ± 0.35 v
1.32 × 105 ± 0.3 per 5 × 106 PBL
plated initially). When compared with non-TNF-supplemented cultures, TNF-treated DC demonstrated an increase in surface expression of CD4, CD80, and CD86, with a concomitant decrease in
CD11c, CD32, and CD33 levels (Fig 2). As
shown in Fig 3, TNF-treated DC also
exhibited high-level expression of CD83, a marker of mature DC, as
reported previously.40 The addition of TNF to
GM-CSF/IL-4-driven DC did not markedly alter the high level of
expression of CD45RO, and these DC remained CD14 and
CD25 (Fig 3). Collectively, these data showed that the
addition of recombinant TNF could enhance the expression of
costimulatory molecules on and increase the number of
GM-CSF/IL-4-driven DC in vitro.
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| Fig 2.
Phenotypic changes in cytokine-driven DC when cultured in
the presence of TNF detected by a panel of antihuman FITC- or
PE-labeled antibodies and FACS analysis. PBMC were cultured in XVIVO-15
serum-free medium containing GM-CSF and IL-4 for 7 days. TNF was
then added at day 7 and the cultures were allowed to proceed for an
additional 7-day period. When compared with non-TNF-supplemented DC
cultures (shaded histograms), the addition of TNF resulted in
positive cell surface expression of CD80 and an upregulated expression of CD86 costimulatory molecules (open histograms). The x-axis is a
logarithmic scale of fluorescence intensity and the y-axis represents
counts.
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| Fig 3.
TNF-treated DC express a high level of CD83. Compared
with non-TNF-supplemented 14-day DC cultures (shaded histograms), the addition of TNF (at day 7) resulted in strong expression of the
mature DC marker, CD83, by FACS analysis (dotted open histograms). Compared with isotype-matched control monoclonal antibody staining, both TNF-treated and non-TNF-treated DC did not show a notable change in high-level CD45RO expression and remained negative for CD14
and CD25 expression. The x-axis is a logarithmic scale of fluorescence
intensity and the y-axis represents counts. Similar results were
obtained in a second repeat experiment.
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TNF enhances the capacity of DC to mediate primary
allo-stimulation of and soluble antigen presentation to T cells.
We next tested DC function in both allogeneic mixed leukocyte response
(MLR) and soluble antigen presentation assays. DC generated in the
presence of GM-CSF and IL-4 with or without TNF were compared. The
number of DC isolated by metrizamide gradient separation that were
added from the two separate cultures (ie, with and without TNF
addition) in the functional assays were equal based on morphology (veiled appearance), size (large), and coexpression of high level CD86
and HLA-DR, but negative for CD14. Different numbers of DC from two
separate donors were cultured with a fixed number of allogeneic PBMC.
As shown in Fig 4, 14-day cultured DC
generated in the presence of TNF (when added at day 7) possessed
enhanced capacity to stimulate a primary allogeneic MLR (ie, a further 8-fold increase in stimulation indices).
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| Fig 4.
TNF-treated DC are more stimulatory than
non-TNF-cultured DC in a primary, 6-day allogeneic mixed leukocyte
reaction. PBMC from two separate donors (B and C) were used to generate
DC; a single PBMC donor (A) served as the source of responder T cells in the assay. Various responder: stimulator (R:S) ratios were included,
as described in the Materials and Methods. DC cultured in the presence
of TNF stimulated greater T-cell proliferative responses at all R:S
ratios tested. The SEM of triplicate wells was always less than 15% of
the mean. Experiments were repeated five times with similar results.
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We then compared TNF-treated versus non-TNF-treated DC for their
capacity to present TT and CAD antigens to autologous CD4+
T cells. As shown in Fig 5, the
TNF-treated DC were considerably more efficient at presenting lower
concentrations of soluble TT and CAD antigen(s) (eg, further 10- to
100-fold increases in stimulation indices) to purified autologous
CD4+ T cells. In addition, at the same dose of antigen(s),
markedly fewer TNF-treated DC were required to trigger specific
proliferative responses of autologous CD4+ T cells (Fig
6). This finding was demonstrated most
clearly at points at which the T-cell response had already plateaued
with increasing numbers of non-TNF-treated DC. By light microscopy, the culture plates of TNF-treated DC displayed a stronger capacity to cluster T cells than those of non-TNF-treated DC (data not shown).
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| Fig 5.
TNF-treated DC possess enhanced antigen-presenting
function. Proliferative responses of purified autologous
CD4+ T cells to different concentrations of soluble
antigens presented by autologous DC were measured at day 6. Non-TNF-treated or TNF-treated DC were pulsed with either TT
(upper) or CAD (lower), as described in the Materials and Methods. The
background cpm of the proliferative response in the absence of antigens
pulsed on DC (ie, unpulsed DC plus CD4+ T cells or the
autologous MLR; upper, +TNF = 5,236 cpm; TNF = 942 cpm; lower, +TNF = 4,102 cpm, TNF = 929 cpm) were
subtracted. Experiments were repeated five times with similar
results.
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| Fig 6.
Fewer numbers of TNF-treated DC can efficiently
present soluble antigens to autologous CD4+ T cells.
Purified CD4+ T cells were cultured with different
numbers of DC generated in the presence or absence of TNF. DC were
pulsed with 12.2 µg/mL TT (upper) or 10 µg/mL CAD (lower), as
described in the Materials and Methods. Proliferative responses were
measured on day 6. Experiments were repeated five times with similar
results.
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TNF treatment of DC reduces but does not eliminate
phagocytic function.
We next tested the effect of exogenous TNF on the phagocytic
function of DC cultured in GM-CSF and IL-4 by evaluating by FACS the
uptake of FITC-labeled dextran particles of differing molecular
weights. The amount of dextran particles incorporated by TNF-treated
DC on a per cell basis was less compared with non-TNF-treated DC as
measured by mean channel fluorescence (Fig 7). However, the overall percentages of
both TNF-treated and non-TNF-treated DC showing uptake of
dextran particles of all three molecular weight sizes were comparable
(~80%) for at least up to the 4 hours of analysis (data not shown).
Phagocytosis was measurable at 37°C but not at 4°C. Thus, the
addition of TNF resulted in DC that were still capable of phagocytic
activity, albeit at a modestly reduced level, compared with DC cultured in the absence of this additional cytokine.
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| Fig 7.
Phagocytosis of FITC-labeled dextran particles by DC
treated with or without TNF. Although TNF-treated DC displayed a
lower degree of phagocytosis on a per cell basis as measured by
fluoresence intensity, the overall percentages of DC capable of dextran
particle uptake were equivalent between DC cultured with or without
TNF. DC were incubated for the indicated periods at 4°C ( ) or
37°C ( ) with FITC-dextran of varying molecular weights. DC were
washed free of unbound particles and were then analyzed by FACS, as
described in the Materials and Methods. Data are expressed as mean
fluorescence channel (MFC) calculated by FACS computer software.
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Removal of TNF from DC cultures reduces its enhancing
effects on the expression of costimulatory molecules and on primary
allo-stimulation of and soluble antigen presentation to T cells.
The effect of removing TNF at different time points during DC
culture was next evaluated. TNF was added to 7-day PBMC cultured in
SFM with GM-CSF and IL-4. Two or 4 days later, cells in these DC
cultures were then washed to remove cytokines and replated in fresh SFM
with GM-CSF and IL-4 for an additional 5 or 3 days, respectively. The
14-day DC cultures were then analyzed for phenotype and T-cell
stimulatory capacity in vitro. When compared with DC cultured in the
presence of TNF for 7 days, removal of TNF resulted in reduced
surface expression of the mature DC marker, CD83, as well as the
costimulatory molecules, CD80 and CD86 (Fig
8). As shown in Table 1, the yield of DC
harvested at day 14 was also reduced after removal of TNF compared
with a DC culture containing TNF for the entire 7-day period (see
experiment no. 8). Figure 9 shows the
effect of early removal of TNF from the DC cultures on stimulation
of antitetanus and primary allogeneic T-cell responses in vitro. When
compared with DC cultured in the presence of TNF for a 7-day period,
removal of TNF from the DC cultures after 2 or 4 days resulted in
significant reductions in both T-cell responses.
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| Fig 8.
Removal of TNF from DC cultures results in reduced
levels of CD80, CD86, and CD83 expression. TNF added to 7-day DC
cultures was subsequently removed by washing the cells 2 and 4 days
later [denoted as TNF day 2() and TNF day 4(), respectively].
The cultures were then replated in fresh SFM with added GM-CSF and IL-4
for an additional 5 or 3 days, respectively. Comparisons were made with
DC cultured without TNF (denoted Non-TNF DC) and those cultured in
TNF for the complete 7-day period [denoted TNF day 7(+)]. FACS
analysis of mean fluorescence channel is depicted. The results are
representative of two experiments.
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| Fig 9.
Removal of TNF from DC cultures results in reduced
levels of antitetanus and primary allogeneic T-cell stimulation in
vitro. Purified CD4+ T cells were cultured with DC
generated in the presence of TNF [denoted TNF DC day 7(+)],
absence of TNF (denoted Non-TNF DC), or after the removal of TNF
from the cultures at 2 days [denoted TNF DC day 2()] or 4 days
[denoted TNF DC day 4()]. Proliferative responses were measured on
day 6 at a DC to T-cell ratio of 1:20. The SEM of triplicate wells was
always less than 15% of the mean. T cells alone = 1,534 ± 186 cpm;
T cells + PHA = 30,081 ± 1,979 cpm. The results are
representative of two experiments.
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| |
DISCUSSION |
Prior studies have identified proliferating progenitors within the
CD34+ cell fraction of human peripheral blood that can be
driven with cytokines, particularly GM-CSF and IL-4, to develop into
potent dendritic cells over a 1- to 2-week period in
culture.25,26,28 DC can also be generated from relatively
small subpopulations of myeloid and lymphoid cells in the presence of
cytokines.17,27,28,30,38,39 Early studies demonstrated that
the combination of GM-CSF and IL-4 could facilitate the generation of
relatively large numbers of dendritic cells from the adherent fraction
of PBMC in media containing FCS.33 Bender et
al41 and Romani et al32 modified the methods to
generate mature DC that involved cytokines combined with special
conditioned medium. In these studies, the DC progenitor population of
cells was first depleted of CD2+ cells, which were later
reported to contain DC precursors.38,39
In the current study, we focused on strategies to generate larger
numbers of functional DC with more potent T-cell stimulatory activity.
Adherent PBMC were first plated in SFM with GM-CSF and IL-4 for 7 days,
followed by the addition of TNF to these cultures for another 7 days. Neither culture was depleted of CD2+ or
CD14+ DC precursors. Seven days of continuous TNF
exposure (added at day 7 of DC culture) was optimal to obtain increased
yield and function of DC. Cells from DC cultures that contained TNF were found to express increased surface expression of the costimulatory molecules CD80 and CD86, as well as the mature DC marker CD83. Earlier
studies by Zhou and Tedder40 had demonstrated that the combination of GM-CSF, IL-4, and TNF could drive the differentiation of CD14+ blood monocytes to mature CD83+ DC
with potent T-cell allostimulatory capacity in vitro.40 We
found that early removal of TNF from DC cultures resulted in a lower
cell yield, a reduced cell surface expression of CD80, CD83, and CD86
molecules, and an inferior capacity to trigger allogeneic and
antitetanus CD4+ T-cell responses. Thus, the continued
presence of TNF (for at least 7 days) in combination with GM-CSF and
IL-4 was necessary for achieving optimum effects on DC. In additional
studies (not shown), removal of TNF from the DC cultures at day 14 did not impact on (ie, reverse) the TNF-induced changes for at least another 2 to 3 days (ie, day-16 or -17 cultures). Beyond this point, in
serum-free medium, the viability of DC decreased in cultures with or
without the addition of TNF. We were able to generate nearly 3 × 105 DC per 5 × 106 PBMC, which was on average
twofold greater than the cultures prepared in the absence of TNF. It
is conceivable that TNF played a role in the maintenance of DC
viability in culture, because it has been reported that this cytokine
can inhibit spontaneous apoptosis of DC in vitro.31 Indeed,
we have observed that DC will undergo apoptosis from day 11 of culture
in SFM with GM-CSF and IL-4 in the absence of TNF (not shown). In
contrast, we have not observed such an apoptotic event in cultures
containing added TNF at least until day 16. Increased numbers of DC
in cultures containing TNF could also be the result of the activity
of this cytokine on certain DC precursor subsets that are not dependent on GM-CSF but would differentiate into DC in the presence of
TNF.42 However, because differences in cell yields were
not apparent until several days after TNF addition (ie, after day 11 of culture), it is believed that lack of apoptosis was the predominant
effect responsible for the increased DC yield in the TNF-containing cultures.
We have compared DC cultured with and without TNF for expression of
certain cell surface molecules, phagocytic activity, and capacity to
stimulate a primary MLR as well as to present defined antigens to
CD4+ responder T cells. We confirmed that adherent PBMC
cultured in SFM containing GM-CSF and IL-4 resulted in cells with
typical DC morphology (veils) and phenotype. These DC also demonstrated efficient uptake of exogenous dextran particles via fluid-phase macropinocytosis and could trigger CD4+ T-cell responses to
TT and CAD antigens in vitro. However, when TNF was added at day 7, the DC produced by day 14 were more stimulatory to T cells and
displayed increased expression of the costimulatory molecules, CD80 and
CD86. These molecules have been shown to play differential roles in
stimulating Th1 versus Th2 cell responses,43 including
those leading to tumor rejection.44 Although we found that
these DC were somewhat reduced in their capacity to engulf dextran
particles on a per cell basis, overall percentages (~80%) of DC that
were positive for particle uptake over a 4-hour time course were
similar between the two cultures. In some instances, TNF had been
shown to have no significant effect on the uptake levels of similar
tracer molecules.36
One of the mechanisms that may contribute to the more efficient
stimulatory capacity of DC generated in the presence of added TNF is
their enhanced capacity for clustering T cells in an
antigen-independent fashion.45 Indeed, we observed in
cultures that TNF-treated DC could mediate a pronounced capacity for
clustering T cells when compared with non-TNF-treated DC (data not
shown). Some investigators reported that TNF decreased the capacity
of antigen processing and presentation of DC.33 The
enhancement of DC function with TNF in our study may be due to the
timing and duration of its addition to the cultures with GM-CSF and
IL-4. It may be considered that TNF has differing effects on certain
stages of DC maturation, because TNF has been shown to
bidirectionally modulate the viability of hematopoietic progenitor
cells in vitro37 and shorter exposure times to this
cytokine were shown to be either inhibitory or have no
effect24,33 (data not shown).
Koch et al46 reported that populations of cultured
Langerhans cells (LC) were not completely inactive in processing and presenting native proteins and that populations of spleen DC obtained by a standard method involving overnight culture were able to process
intact protein to some, albeit lesser, extent. These findings were
attributed to the fact that populations of mature DC are heterogeneous.
Moreover, small numbers of immature DC coexisting within populations of
mature DC, such as cultured LC or spleen DC, could account for the
residual antigen processing activity. The data reported by Sallusto and
Lanzavecchia33 obtained with human DC derived from
monocytes suggest that the interplay of various cytokines may be more
complex. In these studies, TNF induced the downregulation of
processing activity and the upregulation of T-cell allostimulatory
function in human blood-derived DC. This finding would imply that,
rather than being inhibitory, TNF was a crucial trigger for the
process of DC maturation. In our study, 7-day incubation of DC with a
relatively low concentration of TNF improved DC presentation of
soluble TET and CAD antigens at a time when their capacity to capture
dextran particles was depressed on a per cell basis. It is conceivable
that DC cultures remain heterogeneous and that subsets of residual
(partly) immature DC within populations of mature DC were endowed with
biologically significant ability to handle native protein antigen(s).
Several mechanisms have been reported to contribute to the efficient
antigen presentation by DC, including a capacity for clustering T cells
in an antigen-independent fashion45; the expression of high
levels of MHC molecules, allowing presentation of more T-cell
determinants1,2; the high expression of adhesion and
costimulatory molecules and the low surface charge,1,2
which may lower the number of determinants required for T-cell
activation; the high level of fluid-phase pinocytosis47;
and the expression of functional Fc R.48 In addition, DC
have been shown to produce numerous cytokines with potent activity on a
variety of immune cells.16,17 In this regard, we attempted to determine whether there existed differential expression of various
cytokines between TNF-treated and non-TNF-treated DC. For this
analysis, we elected to evaluate cytokines with known T-cell activities
that included activation and proliferation (ie, IL-1, IL-4, IL-6,
IL-7, IL-12, IL-15, interferon-, and GM-CSF). Reverse
transcription-polymerase chain reaction assays performed on FACS-sorted TNF-cultured and non-TNF-cultured DC (>95%
purity) did not show differences in the levels of transcript expression between these two DC groups, although this methodology does not show
actual levels of secreted cytokines (data not shown). On the basis of
the findings from this current study, we have begun to compare in vitro
the capacity of TNF-treated and non-TNF-treated DC to process
and present known tumor peptides3,4,18-20 versus whole
tumor cell lysates5-7 to autologous T cells from advanced cancer patients.
| |
FOOTNOTES |
Submitted September 30, 1997;
accepted February 13, 1998.
Supported by grants from the National Institutes of Health (Grants No.
1 P01 CA59327 and M01-RR00042), from the DOD/USAMRMC (DAMD17-96-1-6103), and from the US Army Research Office
(DAAG55-97-1-0239).
Address reprint requests to James J. Mulé, PhD, Department of
Surgery, University of Michigan Medical Center, 1520c MSRB-1, 1150 W
Medical Center Dr, Ann Arbor, MI 48109-0666.
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 |
The authors thank Dr Satwant Narula, Dr Mary Ellen Rybak, and Chris
DeLuca of Schering-Plough Research Institute for the generous supplies
of recombinant human GM-CSF and IL-4 and thank Dr Douglas Fraker of the
University of Pennsylvania Medical Center for the gift of recombinant
human TNF. We also thank Michelle Walsh of the U-M GCRC and Sandra
Hoffmann of the U-M Blood Bank for leukapheresis samples. The critical
review of the manuscript by Dr Laurence Boxer is greatly appreciated.
| |
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K. Nickerson, T. J. Sisk, N. Inohara, C. S. K. Yee, J. Kennell, M.-C. Cho, P. J. Yannie II, G. Nunez, and C.-H. Chang
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Abstract
Introduction
Abstract