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,
Departments of
* General and Thoracic Surgery, Pathology, Immunology, and
Internal Medicine, and
Center for Genetic and Cellular Therapies, Duke University Medical Center, Durham, North Carolina; and
Immunex Corporation, Seattle, Washington
Correspondence: Michael A. Morse, M.D., Duke University Medical Center, Box 3233, Durham, NC 27710. E-mail: m.morse{at}cgct.duke.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) alone. Instead, we found that
Flt3L-mobilized DC from cancer patients require a sequence of specific
signals for maturation, which included initial treatment with
granulocyte macrophage-colony stimulating factor followed by a
combination of maturation signals such as CD40L and IFN-.
Flt3L-mobilized DC matured in this manner possessed greater T
cell-stimulatory function than nonmatured DC. The ability to generate
phenotypically mature, IL-12-producing DC1 from peripheral blood
mononuclear cells mobilized by Flt3L will have important implications
for the development of effective cancer immunotherapy
strategies.
Key Words: IFN- • CD40 ligand • IL-12 • GM-CSF • immunotherapy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-) and the induction of cytotoxic T lymphocytes (CTL), which are
thought to be effectors for anti-tumor responses. Dendritic cells (DC)
are potent antigen-presenting cells (APC) and are essential for the
initiation of primary T cell-based immune responses. Because of the
potential therapeutic applications of manipulating antigen-specific T
cell responses, agents that may expand the population of circulating DC
are of immense interest. The ligand for the receptor tyrosine kinase
fms-like tyrosine kinase 3 (Flt3L), also referred to as fetal liver
kinase-2, has an important role in the expansion of early hematopoietic
progenitors as well as DC [1
]. These observations lead
to early clinical trials of Flt3L in normal volunteers
[2
] and in cancer patients [3
], in which
4- to 30-fold increases in the number of peripheral blood DC were
reported.
Although the expansion of circulating DC may prove beneficial in
inducing Th1 responses, the immunologic consequences of DC mobilization
in humans may be skewed by the specific subset of DC that are expanded.
Two distinct lineages of DC have been described in humans, DC1 and DC2,
according to their ability to induce naive T cell differentiation to
Th1 and Th2 effector cells, respectively [4
]. We have
found that in humans, Flt3L mobilization leads to DC1 and DC2 expansion
in vivo; however, the majority of Flt3L-mobilized DC was phenotypically
immature [3
]. The immunologic consequences of DC
maturation are as important as lineage, because the immunoregulatory
potential of DC may depend on their state of maturation and activation.
Specifically, immature DC capture and process antigen, and mature DC
shift function to antigen presentation and T cell activation
[5
]. One critical consequence of DC maturation is the
production of interleukin (IL)-12, which plays a pivotal role in the
induction of Th1 responses [6
, 7
]. We have
previously shown that only the combination of signals such as IFN-
and CD40 ligand (CD40L) treatment induces the production of high levels
of IL-12 from a subset of mature, in vitro-generated DC. The
IL-12-producing DC were more effective in inducing tumor
antigen-specific CTL generation in vitro compared with the
non-IL-12-producing DC [8
]. We hypothesized that the
signals that have been shown to induce maturation of ex vivo-generated
DC would be sufficient to mature Flt3L-mobilized DC. Therefore, we
treated cancer patients with hematologically active doses of Flt3L for
14 days. We then harvested peripheral blood mononuclear cells (PBMC) by
leukaphersis and attempted to mature DC in vitro using IFN- and
CD40L. Unexpectedly, we were unable to detect significant IL-12
production from these Flt3L-mobilized DC using CD40L alone or a
combination of CD40L and IFN- treatment. We found that
Flt3L-mobilized DC from cancer patients require initial treatment with
granulocyte macrophage-colony stimulating factor (GM-CSF) followed by
the combination of IFN- and CD40L to generate phenotypically mature,
IL-12-producing DC1. These mature, Flt3L-mobilized DC have greater T
cell-stimulatory activity than nonmatured DC.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation and maturation of DC
All individuals received Flt3L (20 µg/Kg/d sq) for 10 days,
followed within 1 day by leukapheresis. PBMC were isolated by gradient
centrifugation over Ficoll-HypaqueTM Plus (Amersham Pharmacia Biotech
AB, Uppsala, Sweden). PBMC were resuspended in AIM V medium (Gibco-BRL,
Grand Island, NY) at 3 x 106 cells/ml and were plated
polystyrene-culture plates (Costar, Corning, NY). GM-CSF (800 U/ml; R&D
Systems, Minneapolis, MN) was added, except where specified in Results,
and cells were incubated at 37°C with 5%-humidified CO2
for 24 h. Next, one or a combination of the following cytokines
were added as indicated: tumor necrosis factor 
(TNF-; 100 ng/ml;
Endogen, Woburn, MA); CD40 ligand trimer (CD40L; 1 µg/ml; Immunex,
Seattle, WA); IFN-1b (ActimmuneTM; 1000 U/ml; InterMune, Palo Alto,
CA); and/or lipopolysaccharide (LPS; 100 ng/ml; Sigma Chemical Co., St.
Louis, MO). Cells were then incubated for another 24 h,
supernatants were sampled, and cells were subject to further analysis.
IL-12 enzyme-linked immunosorbent assay (ELISA)
For the detection of IL-12 production by Flt3L-mobilized DC,
ELISA was performed on 24-h culture supernatants using the QuantikineTM
IL-12 HS ELISA kit (R&D Systems), according to the manufacturer’s
instructions.
Intracellular IL-12 analysis and phenotypic analysis by flow
cytometry
After maturation of cells in the indicated cytokine-supplemented
media, 10 µg/ml brefeldin A (Sigma Chemical Co.) was added to each
well to inhibit cytokine secretion. DC were incubated for 4 h,
harvested using cell-dissociation buffer (Gibco-BRL), and then placed
on ice until all samples were harvested. Cells were washed with 5 ml
Dulbecco’s phosphate-buffered saline (DPBS; Gibco-BRL) and were fixed
with 1% formaldehyde (Sigma Chemical Co.) and 1% bovine serum albumin
(Sigma Chemical Co.) in PBS for 10 min at room temperature (RT). Cells
were then permeabilized with 0.5% saponin (Sigma Chemical Co.) in PBS
for 20 min at 37°C followed by vortexing. Samples were washed and
stained with one or more of the following monoclonal antibodies (mAb):
anti-CD14-fluorescein isothiocyanate (FITC) or -PerCP, anti-CD19-FITC,
anti-CD3-FITC, anti-CD80-phycoerythrin (PE), and anti-human leukocyte
antigen (HLA)-DR-PerCP (all from Becton Dickinson, San Jose, CA);
anti-CD83-FITC (Immunotech, Marseille, France) or -PE (Becton
Dickinson); anti-CD11c-FITC (Dako, Carpenteria, CA) or -APC (Becton
Dickinson); anti-CXCR4 (PharMingen, San Diego, CA); and anti-CCR5
(PharMingen). In some experiments, the cells were stained with these
antibodies first and then fixed and permeabilized as described (results
were equivalent). Cells were washed with PBS containing 2% fetal calf
serum (FCS). Anti-human IL-12 (p40/p70)-PE or -APC (PharMingen) was
added to the cells, and they were incubated for 20 min at RT in the
dark, washed two times with PBS containing 2% FCS, and fixed with 1%
paraformaldehyde. Isotype-matched negative controls were used, and
gates were set to include only 1% of the counts from the negative
control. Labeled cells were analyzed on a FACSCaliber (Becton Dickinson
Biosciences) flow cytometer using Cell Quest software.
For detection of CCR7, the cells were incubated with anti-CCR7 (PharMingen) for 20 min at RT and washed twice with PBS containing 2% FCS. Biotinylated rat anti-mouse immunoglobulin M (IgM; R6-60.2, Southern Biotechnology Associates, Inc., Birmingham, AL) was added to the cells and incubated for 20 min at RT. Cells were washed two times with PBS containg 2% FCS. Streptavidin-PE (PharMingen) was added to the cells and incubated for 20 min at RT. Cells were washed two times with PBS containing 2% FCS and fixed with 1% paraformaldehyde. Permeabilization of cells was performed using 0.5% saponin (Sigma Chemical Co.) in PBS for 20 min at 37°C. Cells were washed with PBS containing 2% FCS. Anti-IL-12 p40/p70 was added to the cells and incubated for 20 min at RT, washed two times with PBS containing 2% FCS, and fixed with 1% paraformaldehyde prior to analysis.
To detect DC-SIGN, anti-DC-SIGN (DC4) and anti-DC-SIGN (DC28; provided by the NIH AIDS Research and Reference Reagent Program, Rockville, MD) were added to cells for 20 min and washed as described above. Goat anti-mouse Ig-PE [IgM+IgG+IgA(H+L); Southern Biotechnology Associates, Inc.] was added, and the cells were incubated for 20 min and washed two times. Cells were incubated with 1% mouse serum in PBS for 20 min prior to staining with any other fluorescently labeled antibodies as previously described. Cells were washed, fixed, permeabilized, and stained for IL-12 p40/p70 as described above.
Data were analyzed by gating on the DC (large CD11c+/CD14- cell or large, lineage (CD3, CD14, CD19)-negative, HLA-DR-positive cells). These cells were then analyzed for IL-12 expression, and a panel of the other markers was described above. Gated events (1–5x104) were analyzed for each sample.
Detection of allo-lymphocyte stimulation by enzyme-linked
immunospot (ELISPOT)
Multiscreen-HA 96-well plates (Millipore, Bedford, MA) were
coated overnight at 4°C with 100 µl/well of 10 mg/ml mouse
anti-human IFN- mAb (diaPharma Group, Inc., West Chester, OH)
in DPBS (Life Technologies, Gaithersburg, MD). The plates were washed
three times for 5 min each with 150 ml DPBS/well and were blocked with
200 µl/well RPMI 1640, 10% HuAB serum, 25 mM HEPES, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 2 mM glutamine for 1 h
at 37°C in 5% CO2. Flt3L-mobilized PBMC cultured with or
without CD40L and IFN- (1x105 cells/well) and 1 x
105 allogeneic oligo-polyclonal tumor-infiltrating
lymphocytes (TILs; kindly provided by Steven A. Rosenberg, NCI,
Bethesda, MD) were added to each well as responders for 18–20 h at
37°C in 5% CO2. The plates were washed with 0.05%
Tween/DPBS using the Tecan 96PW plate washer. Mouse anti-human
IFN--biotinylated mAb (diaPharma Group, Inc.), 100 µl at 1 µg/ml
in DPBS, was added to each well, and the plates were incubated for
2 h at 37°C, 5% CO2. Vectastain ABC Peroxidase
(Vector Labs, Inc., Burlingame, CA) was added at 100 µl/well for
1 h at RT after washing. The plates were washed for a final time
with 0.05% Tween/DPBS followed by DPBS. Color was developed using 100
µl/well 3-amino-9-ethyl-carbazole (Sigma Chemical Co.), reconstituted
in an acetate buffer for 4 min at RT in the dark. Color development was
stopped with deionized water. Basins were removed, and the membranes
were dried overnight in the dark. Membranes were removed using sealing
tape (Millipore). Membranes were read by Zellnet Consulting, Inc. (New
York, NY) using the KS ELISPOT Automated Reader system with the KS
ELISPOT 4.2 software (Carl Zeiss, Inc., Thornwood, NY) to determine the
number of spots/well. One spot represents one cytokine-secreting cell.
IL-12 p40/70 ELISPOT
IL-12 ELISPOT was performed using the human IL-12 (total IL-12)
ELISPOT kit (Mabtech, Sweden). All reagents not provided in the Mabtech
kit were the same as the reagents used in the IFN- ELISPOT mentioned
above. Flt3L-mobilized PBMC were cultured with or without CD40L and
IFN- and were plated in sextuplicate at 2 x 105
cells/well and incubated for 18–20 h at 37°C in 5% CO2.
Graphical and statistical analysis
Microsoft Excel was used for graphical and statistical analyses
of data where applicable. Flow cytometric data were analyzed using the
paired Student’s t-test with a Bonferroni correction.
P < .05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and
CD40L increases the percentage of CD11c+CD14- cells are quite effective in maturing myeloid DC generated in
vitro, but it is not expected that these cytokines would increase the
percentage of DC. Rissoan et al. [4
] had reported high
levels of GM-CSF receptors on human monocyte-derived DC1 subsets. We
therefore included GM-CSF in the culture for 18–24 h prior to the
addition of CD40L and IFN- in an attempt to expand the DC1 subset.
As illustrated in Figure 1
, the percentage of CD11c+CD14- expression among large cells
increases markedly following treatment of Flt3L-mobilized PBMC with
GM-CSF followed by CD40L and IFN-. Similar results were seen in
cultures from each of three cancer patients who underwent treatment
with Flt3L. These data suggest that GM-CSF increases the transition of
some CD11c+CD14+ monocytoid cells into DC1.
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or CD40L results
in an increase in CD80 and CD83 expression, but the CD80 is relatively
dim. The combination of GM-CSF followed by CD40L and IFN- enhances
expression of CD80 and CD83 even further, and the expression of CD80 is
brighter. To determine whether these results are unique to the
Flt3L-mobilized DC of cancer patients, we also evaluated expression of
CD80 and CD83 on the matured DC of healthy volunteers who had received
Flt3L. It is interesting that for three donors repeated twice, the
viability of the DC matured for 2 days without the presence of GM-CSF
was extremely poor (<20%), although there was a suggestion of some
CD83 and CD80 expression on the viable cells (data not shown). Only
when GM-CSF was provided in the culture media prior to the addition of
CD40L and IFN- was there viability >80% and obvious up-regulation
of CD80 and CD83 (see data shown in Fig. 7
).
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in
vitro induces IL-12 p70 production and CD40L,
we also wanted to demonstrate functional maturation as defined by the
production of IL-12. Flt3L-mobilized PBMC obtained from each of three
patients were incubated in AIM V medium with GM-CSF followed by various
combinations of agents previously described to mature DC, including
CD40L, IFN-, and LPS. After the 48-h incubation, a sample of the
supernatant was removed for determination of bulk IL-12 p70
concentration by ELISA (Fig. 3
). Baseline levels (approximately 10 pg/ml) of IL-12 p70 production
were noted in PBMC incubated in AIM V alone, with GM-CSF alone, with
TNF- alone, and with GM-CSF and IFN-. We saw no significant
increase in IL-12 production with CD40L and IFN- treatment. In stark
contrast, when GM-CSF was followed by CD40L and IFN-, significantly
greater levels of IL-12 production were observed.
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in
vitro results in high-level IL-12 production by a subset of DC are functionally mature, we wanted to
determine the phenotype of the cells actually producing the IL-12 using
intracellular IL-12 detection. We observed that only the CD11c+CD14-
DC produced IL-12 (Fig. 4
). In Flt3L-mobilized PBMC from cancer patients, the vast majority
of IL-12-producing large cells was found to be CD11c+CD14-
(90.9±8.5%, n=4). In addition, the IL-12-producing DC are
CD83+, although a minority of CD83+ cells express IL-12. The greatest
percentage of DC-producing IL-12 occurred with GM-CSF followed by
IFN- and CD40L (Fig. 5
). This intracellular IL-12 p40/p70 data parallel the IL-12 p70
data from the ELISA. Similar results were observed for the
Flt3L-mobilized DC of healthy volunteers as well (data not shown).
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, and CD40L
produced IL-12 compared with less than 0.05% of nonmatured cells
(Fig. 6
). Because DC account for 5–20% of the PBMC, this suggests that
2.5–10% of DC produce IL-12 as determined by this method, in
agreement with estimates by flow cytometric analysis of IL-12-producing
DC.
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and also the matured DC of healthy volunteers (Fig. 7C)
.
Maturation led to up-regulation of CD80, CD83, mannose receptor, and
CCR7 and down-regulation of CCR5. There was little change in CXCR4, DC
SIGN28, and DC SIGN 4. We observed that the IL-12-secreting DC were
those that were high expressors of CD80, CD83, and mannose receptor but
lower (or negative) expressors of CXCR4, CCR5, CCR7, and DC-SIGN.
Qualitatively, the data for the healthy volunteer DC were similar for
the markers evaluated. There was considerable variability from person
to person in the degree of brightness of many of the markers, which we
believe accounts for the apparent difference in CXCR4 and CCR7. These
data confirm that the IL-12-secreting DC are phenotypically consistent
with mature DC and that cancer patient DC have a similar phenotype to
healthy, volunteer DC, allowing for person-to-person variability.
Function of matured DC
One characteristic of functionally matured DC is greater
allostimulatory activity. To compare the immune function of the
matured, Flt3L-mobilized DC with nonmatured DC, we performed a mixed
leukocyte ELISPOT assay in which stimulation of allogeneic T cells by
DC was measured by cytokine production from the T cells in response to
co-culture with Flt3L-mobilized DC. Significantly greater numbers of T
cells were stimulated to produce IFN- by matured DC (Fig. 8
). This IFN- was produced by the T cells, as DC alone produced
no IFN-. These data suggest that the GM-CSF/CD40L/ IFN--treated
cells are functionally mature.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
and T cell-derived signals (CD40L) [10
11
12
]. Mature DC
have been characterized by phenotypic markers such as up-regulation of
CD80 and CD83 and by functional changes such as greater allostimulatory
activity in mixed leukocyte assays, but physiologically, the most
important characteristic is the ability to produce IL-12, which
polarizes Th cells toward Th1 responses. We have previously
demonstrated that IL-12 production defines the functionally mature DC
amongst those myeloid DC generated in vitro with GM-CSF and IL-4 and
exposed to CD40L and IFN- [8
]. In the present paper,
we extend these observations to the blood DC1 mobilized with Flt3L, but
we report that the addition of GM-CSF for 16–24 h prior to the
addition of CD40L and IFN- is required for IL-12 production. This is
not unique to the DC of cancer patients, as a similar requirement was
found for the Flt3L-mobilized DC of healthy volunteers. As
previously reported for in vitro-generated DC, we demonstrate that
only a subset of CD11c+CD14-CD83+ Flt3L-mobilized DC produces IL-12.
As expected, GM-CSF/CD40L/IFN--matured DC possessed greater
allostimulatory function, and the stimulated T cells produced IFN-,
indicating a Th1 polarization.
The requirement for GM-CSF in the maturation strategy described herein
is not unexpected. When GM-CSF was added to murine bone marrow cells at
concentrations similar to those used in our study, mature DC could be
generated following the addition of LPS or TNF- [13
],
but as the GM-CSF was titrated down to very low levels, DC became
resistant to maturation. The need for a day of GM-CSF before adding
CD40L and IFN- is suggested by other work by the same group in which
the addition of LPS on the first day of bone marrow cell culture along
with the GM-CSF resulted in only immature DC [14
].
IL-12 p70 is the functionally active form of IL-12, but we were not able to detect intracellular IL-12 p70 with an available p70 antibody. Therefore, we used an antibody that detects IL-12 p70 and the p40 subunit. Previously, we have shown that IL-12 p40/p70 detected by intracellular cytokine staining parallels IL-12 p70 levels detected in supernatants by ELISA in matured, monocyte-derived DC [8 ]. In the current study, we again confirm by two assays, the IL-12 ELISPOT and IL-12 ELISA, that intracellular IL-12 detected by the p40/p70 antibody parallels production of bioactive IL-12.
The phenotype of the IL-12-secreting, Flt3L-mobilized DC is also generally as expected with some minor differences. DC up-regulate CD80, CD83, and CCR7 [15 ] during maturation and down-regulate CCR5 [15 ]. DC-SIGN has been found on mature and immature in vitro-generated DC, but the fluorescence intensity on mature DC is reported to be half that of immature DC [16 ]. We observed considerable variability from person to person among cancer patients and healthy volunteers regarding expression of these markers. Nonetheless, we also observed CD80 and CD83 up-regulation in cancer patients and healthy volunteers with maturation. Cancer patients and some healthy volunteers had up-regulation of CCR7. DC-SIGN expression was qualitatively similar. Surprisingly, the IL-12 expression did not always occur from the DC with the brightest maturation markers. Although IL-12 was produced by a subset of the brighter CD83- and CD80-positive DC, it was produced from the lower or negatively expressing CXCR4, despite the fact that CXCR4 has been reported to be up-regulated with maturation [15 ]. We do not have an explanation for this finding, but it does suggest that functional maturation as indicated by IL-12 production does not overlap exactly with the entire population of DC deemed phenotypically mature.
In summary, we have demonstrated the requirements for generating
functionally mature DC from Flt3L-mobilized PBMC. The ability to induce
potent T cell stimulation suggests that the mobilized DC matured with
GM-CSF, followed by the combination of CD40L and IFN-, may be useful
as a potent platform for stimulating antigen-specific immune responses
in clinical trials of active immunotherapy.
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ACKNOWLEDGEMENTS |
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Received November 14, 2001; revised April 18, 2002; accepted April 29, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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