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Prepublished online as a Blood First Edition Paper on August 8, 2002; DOI 10.1182/blood-2002-04-1063.
NEOPLASIA
From the University of Minnesota Cancer Center and
Department of Pediatrics, Division of Pediatric Hematology/Oncology and
Blood & Marrow Transplant, Minneapolis; the Department of Veteran
Affairs Medical Center and the Department of Internal Medicine,
University of Iowa College of Medicine, Iowa City; the Coley
Pharmaceutical Group, Wellesley, MA; and the Departments of
Immunobiology and Discovery Research, Immunex Corporation, Seattle, WA.
Bone marrow (BM)-derived dendritic cells (DCs) cultured in
granulocyte-macrophage colony-stimulating factor (GM-CSF) and
interleukin 4 (IL-4) have been used to generate antitumor immune
responses. The cytokine Flt3 ligand (Flt3L) also has been shown to
generate BM DCs. We sought to determine if DCs generated by using Flt3L then matured with lipopolysaccharide (LPS) could lead to DCs with in
vivo anti-acute myelogenous leukemia (anti-AML) activity. LPS and tumor necrosis factor Dendritic cells (DCs) are the most potent
antigen-presenting cell (APC) and have been used to present
tumor-specific antigens as tumor vaccines.1-9 DC vaccines
are being tested as a means of providing antitumor immune response in
patients.4-9 The maturational state of DCs affects the
ability of the cells to uptake, process, and present tumor antigens;
migrate to lymph nodes that drain tumor sites; and ultimately stimulate
a potent T-cell-mediated antitumor response.10 Immature
DCs more effectively capture and process antigen than mature DCs. In
addition, immature DCs appear to more effectively travel to lymph nodes
where, having undergone maturation and up-regulation of costimulatory
molecules in vivo, these now mature DCs can generate a T-cell
response.10-15 The maturational stage of the DCs
ultimately may have significant effects on their ability to function as
vehicles for antitumor immunotherapy.
Different methods have been described for the ex vivo generation of
mature DCs from bone marrow (BM), peripheral blood, or splenic
progenitor cells. Granulocyte-macrophage colony-stimulating factor
(GM-CSF) and interleukin 4 (IL-4) are commonly used together to
generate immature DCs from both murine and human progenitor cells.16 The in vivo administration of the cytokine Flt3
ligand (Flt3L) has been shown to stimulate the proliferation of
hematopoietic progenitors in mice, primates, and
humans.17,18 It also greatly increases the numbers of
functionally active DCs in BM, gastrointestinal lymphoid tissue, liver,
lymph nodes, lung, peripheral blood, peritoneal cavity, spleen, and
thymus.19,20 We have shown that Flt3L has potent
antileukemia effects when administered in vivo as a single agent to
naive mice and mice receiving bone marrow transplants.21 Brasel et al22 described the use of Flt3L to generate DCs
from BM progenitors by culturing with the cytokine for 8 days. These DCs expressed high levels of costimulatory and major histocompatibility complex (MHC) molecules, stimulated a potent allogeneic T-cell response, and were able to process and present protein antigen to
antigen-specific CD4+ T cells. These data suggest
that the use of Flt3L in BM progenitor cultures may be a more
simplified and potent method of generating potent DCs ex vivo. The in
vivo function of Flt3L-generated DCs has not been previously reported.
Cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpGs) are
synthetic oligodeoxynucleotides that mimic immunostimulatory bacterial DNA.23,24 They are unmethylated DNA sequences
containing characteristic CpG dinucleotides in particular base
contexts.23-25 These CpG motifs are thought to be
responsible for initiating a potent immune response in mice, primates,
and humans.26-28 CpGs have been shown to activate APCs
(particularly tissue DCs), leading to up-regulation of costimulatory
molecules necessary for T-cell activation and conversion to effector
cytotoxic T lymphocytes (CTLs), and CpGs also stimulate DC secretion of
proinflammatory cytokines, particularly interferon- Previously, we have shown that TNF- Mice
Cells and cell culture reagents
Generation of DCs from BM progenitors BM was harvested from the long bones of the femur, tibia, and fibula of mice as previously described.36 Red cells were lysed by ammonium chloride incubation, and the single cell suspension was depleted of mature T cells, B cells, granulocytes, and IAb+ (MHC II+) cells by using the following antibody cocktail followed by complement lysis. The antibody cocktail contained the following antibodies: anti-Thy 1.2 (30-H-12), anti-B220 (RA3-6B2), anti-Gr-1 (RA-8C5), and IAb (AF6-120.1.2; American Type Culture Collection [ATCC], Manassas, VA). The DC progenitors were then incubated at 1 × 106 cells/mL Dulbecco modified Eagle medium (DMEM)-complete media with cytokine for 5 to 7 days at 37°C and 10% CO2 in 6-well plates with 3 mL per well. Four distinct cytokine combinations were used to generate DCs from marrow precursors. The first combination used GM-CSF (R&D Systems, Minneapolis, MN) 1000 U/mL and IL-4 (Shering-Plough, Kenilworth, NJ) 1000 U/mL for 5 days followed by 2 further days in culture with TNF- (4 ng/mL; R&D Systems) according
to our previously published studies20 (method 1). On the
basis of literature,37 we chose a second approach to generate mature DCs by using GM-CSF (R&D Systems) 150 U/mL and IL-4
(Shering-Plough) 75 U/mL for 7 days followed by 2 further days in
culture with LPS (0.1 µg/mL; Sigma, St Louis, MO) (method 2), or CpG
oligodeoxynucleotide (CpG 2006; 2 µg/mL) (Coley Pharmaceutical Group,
Wellesley, MA) (method 3). CpG 2006 has been previously shown to
effectively mature human peripheral blood progenitors into
DCs.32 Cytokines were replenished on day 3-4 by removing two thirds of the media and replenishing with fresh media supplemented with cytokine. Nonadherent and loosely adherent cells were removed by
pipetting on day 4-5 and replated with fresh cytokine-containing media
in new 6-well plates. Cells were again replated with fresh cytokine-containing media when the maturational cytokine (TNF-, LPS,
or CPG) was added on days 7 to 9. DCs generated using GM-CSF and IL-4
and matured with TNF- were compared in parallel cultures using the 2 concentrations of GM-CSF and IL-4; there were no differences in the
morphology and phenotype of the cells either before or after TNF-
(data not shown).
For the fourth approach, Flt3L was used as a single cytokine to generate immature DCs. BM progenitors depleted of red blood cells but not mature cells were incubated with Flt3L (200 ng/mL; Immunex, Seattle, WA) for 9 days. Final maturation was accomplished by the addition of LPS (1 µg/mL; Sigma) for the final 24 hours of culture as previously described 22 (method 4). Phenotypic evaluation of DCs The cell surface antigen expression of the DCs was compared by using flow cytometry of cells on days 7 to 9 of incubation after DCs were pulsed with tumor antigen. There was no phenotypic difference detected between DCs evaluated prior to pulsing with tumor lysate and after an 18-hour incubation with lysate (data not shown). Cells were washed and incubated with-FCR (CD16/CD32) (2.4G2) (Pharmingen, San
Diego, CA) at 4°C for 10 minutes to block nonspecific binding of
fluorochromes. The following directly conjugated antibodies
(Pharmingen) were incubated with DCs at 4°C for 30 minutes:
CD8-fluorescein isothiocyanate (FITC), CD4-FITC,
NK1.1-phycoerythrin (PE), B220-PE, CD11b-PE, CD80-FITC,
CD86-FITC, CD-40-FITC, H2Kb-PE, IAb-FITC, and
CD11c-PE. Cells were washed and analyzed using the FACS Caliber
(Becton-Dickinson, San Diego, CA) and were gated and analyzed using
forward and side-scatter plots on 10 000 live events.
Allogeneic T-cell response The ability of the DCs to generate a potent allogeneic T-cell stimulatory response was evaluated using BALB/c T cells isolated from the lymph nodes of normal mice. The T cells were plated at 2 × 105 cells per well and stimulated with graded concentrations of irradiated (30 Gy, Cs source) DCs in 96-well plates. Cells were labeled with tritiated thymidine (1 µCi/well [0.037 MBq/well]) for approximately 18 hours prior to harvesting. Cells were harvested on days 1 to 6, and tritiated thymidine incorporation was calculated as counts per minute with the use of a Beta Counter (Packard Instruments, Downers Grove, IL) in the absence of scintillant application that typically yields counts per minute of 5% to 10% of those with scintillant. Supernatant was obtained from cell cultures prior to the addition of tritiated thymidine and analyzed for Th1 (IL-2, IFN- ) and Th2 (IL-10) cytokines by enzyme-linked
immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN). The
sensitivity of the assay was 3.0 pg/mL for IL-2, 2.0 pg/mL for IFN-,
and 4 pg/mL for IL-10.
In vivo AML protection DCs were generated as described and pulsed with C1498 lysate at a ratio of 3:1 (lysate cell equivalents/DC) on days 7 to 9 of culture and incubated in 6-well plates for 18 hours. DCs were washed 3 times in PBS and resuspended in sterile PBS for injection into mice. Mice (n = 5-10) were vaccinated 14 and 7 days prior to tumor challenge with 0.5 × 106 DCs that were injected into the lateral tail vein at a DC dose and schedule previously determined to provide a high degree of protection to a uniformly lethal dose of C1498 (1-2 × 105 cells). Mice were then challenged on day 0 with a supralethal dose of C1498 tumor cells (1-2 × 106 cells per mouse) injected intravenously to facilitate comparisons of the degree of AML protection.In vivo CTLp generation The cytotoxic T-lymphocyte precursor (CTLp) frequency was analyzed by limiting dilution as previously described.38 Spleens from mice (n = 3 per group) that received DCs and nonvaccinated controls were harvested on day 0, a single cell suspension was obtained by passing the cells through a wire mesh, and red cells were lysed by using ammonium chloride. Graded concentrations of splenocytes (1 × 105 3 × 102) (30 replicates per dilution) were stimulated with 1 × 104
irradiated (100 Gy, Cs source) C1498 tumor cells per well and IL-2 (20 U/mL; Amgen, Thousand Oaks, CA) in 96-well plates for 7 days. Tritiated
thymidine-labeled C1498 tumor cells (1 × 104 per well)
were then added to the cultures and incubated for 4 hours at 37°C.
Parallel dilutions were compared for each group with the irrelevant
tritium-labeled tumor targets YAC-1 (H2a) lymphoma cells
and B16F10 (H2b) melanoma cells. Cytotoxicity was assessed
by the JAM assay39 that measures the level of tritium
retained by cells that have not undergone lysis. The CTLp frequency was
calculated as previously described by means of Poisson-distribution
statistics.40 Results are expressed as the CTLp frequency
per spleen, which takes into account the variability in spleen size
with vaccination.
Statistics The Kaplan-Meier product-limit method was used to calculate survival rates. Differences between groups were determined using the generalized Wilcoxon test. Survival data are also presented as median survival time (MST), the point in time at which half of the mice remain alive.
Similar morphology and phenotype of DCs generated using GM-CSF/IL-4
matured with LPS, CpG, or TNF- , a protocol also used for human
DC generation in clinical trials, provided substantial anti-AML protective responses in naive and BM transplant-treated
mice.21 Labeur et al,37 using a murine model
of squamous cell carcinoma, suggested that DCs matured with LPS were
more potent in vivo, produced higher levels of IL-12, and led to
greater CTL responses compared with DCs matured with TNF-. Comparing
TNF--matured DCs to CpG 1826-matured DCs, Brunner et
al41 showed that DCs matured with this CpG had
superior IL-12 production, greater induction of alloreactive T-cell
responses, and improved in vivo antitumor effects in a murine model of
colon cancer. Because LPS maturation cannot be used clinically owing to
its potential severe systemic toxicities, we sought to determine
whether CpG may provide similar DC maturation properties and provide
comparable or superior in vivo anti-AML responses compared with those
already observed with TNF--matured DCs. Using a different approach,
Brasel et al22 described the in vitro generation of DCs
using Flt3L and LPS. Therefore, we sought to determine if these
Flt3L-generated DCs had similar anti-AML effects in vivo.
Comparing the several methods of DC generation from murine BM precursor
cells, all 4 distinct methods generated cells with morphology
consistent with that described for DCs with large dendrite projections
(data not shown). Consistent with our previous data,22 the
DCs generated using Flt3L/LPS were smaller and had fewer dendrites compared with DCs generated using GM-CSF/IL-4 and matured with TNF-
GM-CSF/IL-4- and Flt3L-generated DCs stimulate potent alloreactive
responses that are comparable with TNF- produced a potent allogeneic T-cell response that
peaked on day 5 and was 5.5-fold higher than spleen cell control
responses. This response was comparable to the alloreactive response of
DCs generated using GM-CSF/IL-4 and matured with either LPS or CpG.
Whereas DCs generated using Flt3L/LPS generated a strong allogeneic
T-cell response, this response was weaker and less sustained compared
with the response observed with the DCs generated using GM-CSF/IL-4 and matured with TNF-, LPS, or CpG.
Because Flt3L-generated DCs have a greater population of
CD8 DCs generated using GM-CSF/IL-4 and matured with TNF- , and pulsed with AML lysate
provide substantial antitumor protection when administered to naive
mice or to mice receiving BM transplants 14 and 7 days prior to a
uniformly lethal C1498 dose (1-2 × 105).21
Labeur et al37 suggested that DCs matured with LPS provide greater in vivo antitumor protection compared with DCs matured with
TNF-. For in vivo comparative studies, a supralethal dose (2 × 106 cells/mouse) of C1498 cells was administered.
The minimal lethal dose of C1498 is 1 × 105 cells per
mouse. We chose a tumor dose more than 10-fold higher than is the
minimal required to be able to clearly discern any differences in
protective effect that may not be evident at a lower tumor cell dose.
Aggregate data from 2 experiments indicate that the cohorts of mice
vaccinated with DCs matured with TNF-
DCs matured with CpGs provide superior in vivo anti-AML protective response compared with DCs generated with GM-CSF/IL-4 and matured with LPS Because the MST of mice that received GM-CSF/IL-4, LPS-matured DCs was longer than mice that received DCs matured with TNF-, we chose
to directly compare LPS-induced DC maturation to that using CpG for in
vivo protective responses. Pooled data from 2 replicate experiments
indicated that DCs generated using GM-CSF/IL-4, then matured with CpG
2006, and pulsed with C1498 lysate provided a significant survival
advantage compared with control mice after lethal tumor challenge (20%
compared with 0%, P  .0001; Figure 4). The survival advantage provided by
DCs matured with CpG was significantly improved compared with the
survival of mice administered GM-CSF/IL-4/LPS-matured DCs (20% versus
13% survival, respectively, P = .02). The MST of mice
that received CpG-matured DCs was longer than mice that received
LPS-matured DCs (73.9 versus 49.98 days, respectively). These results
indicate that CpG-matured DCs provide superior antitumor responses to
LPS-matured DCs in vivo and prolong the MST. Because LPS- and
TNF--matured DCs provide similar antitumor response, we conclude
that CpG-matured DCs are superior to either LPS- or TNF--matured
DCs for providing in vivo antitumor protective responses.
Flt3L generates DCs capable of providing anti-AML protective response To determine whether Flt3L-generated, LPS-matured DCs could provide anti-AML protection when pulsed with C1498 lysate, mice were administered this DC population 14 and 7 days prior to lethal (1 × 106) tumor challenge. Aggregate data from 2 replicate experiments indicated that mice that received the Flt3L/LPS-generated DCs had significantly longer survival compared with nonvaccinated mice after tumor challenge (11% versus 0% surviving, respectively, P < .0001; Figure 5) with MSTs of 36 and 26.3 days, respectively. Moreover, mice that received the GM-CSF/IL-4-generated DCs had significantly improved survival compared with mice that received DCs generated using Flt3L (35% versus 11% survival, respectively, P = .002) with MSTs of 56 and 36 days, respectively. These data indicate that DCs generated by either GM-CSF/IL-4 or Flt3L are capable of providing tumor protection to murine AML, but DCs cultured with GM-CSF/IL-4 are superior to cells cultured with Flt3L in this model system.
All DC-treated mice generated tumor-specific CTLps in response to vaccination Because all cohorts of mice that received DCs prior to tumor challenge had improved survival compared with controls, we determined if there was evidence that a tumor-specific response was achieved in vivo at the time of systemic AML challenge. Tumor-specific CTLps were detectable in all mice that received DC vaccinations (Figure 6). All cohorts of DC-treated mice had an increase in the absolute number of tumor-specific splenic CTLps compared with naive mice. There were no substantial differences among the 4 different propagation methods. These responses were specific for C1498, as there was minimal response to the irrelevant tumor targets B16 and Yac-1. The lack of response to Yac-1 cells indicates that the responding cells were not natural killer (NK) cells, as Yac-1 is sensitive to NK killing.
Our study is the first comparative study evaluating the phenotype
and in vivo biologic function of GM-CSF/IL-4-generated, TNF- CpGs have been shown to induce potent DC-mediated responses when these
agents have been given in vivo.32,33 We have
previously shown that the systemic administration of CpG as a single
agent leads to both protective and therapeutic anti-AML
responses.21 The effect of CpGs on activating DCs leads to
up-regulation of costimulatory molecules on the surface of DCs and
increased cytokine production that further enhances the ability of DCs
to stimulate T-cell responses. Hartmann et al32 have
reported that CpGs are able to generate and mature DCs from human
peripheral blood progenitors. Others have shown that DCs can
be generated by using murine endothelial progenitors in the
presence of CpGs that then leads to DCs with the characteristics of
tissue Langerhans cell.33,34 Our data show that DCs
cultured with GM-CSF/IL-4 and matured with CpG leads to comparable
costimulatory molecule expression to that seen on DCs matured with
LPS and TNF- LPS and CpG mediate their effects on DC activation via binding to
distinct Toll-like receptors (TLRs) on the surface of the DC
(specifically TLR4 and TLR9, respectively).43,44 The exact downstream signaling responses are not clearly understood, but both use
the MyD88 adapter protein for signaling and up-regulation of
NF Flt3L is an effective cytokine for generating immature DCs in vivo and
has been used to generate antitumor immune responses in a variety of
tumor models.53-57 In human phase I clinical trials, the
administration of Flt3L has led to substantial increases in peripheral
blood monocytes and circulating DCs, resulting in increased DCs at
tumor sites as well as increased DCs available for leukapheresis and
vaccine generation.58,59 Clinical and cellular antitumor responses have been observed in up to 50% of the patients treated with
Flt3L-generated DC vaccines.59 Because immature DCs are thought not to be as efficient at presenting antigen to T cells, it may
be more beneficial to generate immature DCs ex vivo using Flt3L and
mature them to obtain maximal antitumor responses. Brasel et
al22 described the use of Flt3L, as a single cytokine, for the ex vivo generation of BM-derived DCs. We have used this method and
further expanded the data by using in vivo studies in a murine model of
AML. Our flow cytometry data are consistent with that of Brasel et
al,22 indicating a population of DCs that coexpress CD8 Several studies have been published on the use of DC vaccines for the
protection from recurrence and treatment of solid
tumors.1-9 We have previously published the successful
prevention of AML after bone marrow transplantation by using DCs pulsed
with AML lysate.21 These previous studies used DCs
generated using GM-CSF/IL-4 and matured with TNF- In conclusion, LPS has been used to mature DCs in vitro and is a potent
stimulator of immune responses in vivo. The clinical use of LPS in
humans is limited because of its risk for inducing substantial
toxicity. Our data indicate that DCs generated using GM-CSF/IL-4 and
CpG produce DCs that are comparable to DCs generated using LPS or
TNF-
We thank Micki Diers and John Vaala for their technical assistance.
Submitted April 4, 2002; accepted July 15, 2002.
Prepublished online as Blood First Edition Paper, August 8, 2002; DOI 10.1182/blood-2002-04-1063.
Supported in part by grants from the Children's Cancer Research Fund, Viking Children's Fund, Coley Pharmaceutical Group, and grants R01 CA-72669 and RO1 CA-85922 from the National Institutes of Health.
A.M.K. has a commercial interest in the Coley Pharmaceutical Group and the CpGs used are products of Coley Pharmaceutical Inc. K.B. has a commercial interest in the Immunex Corporation and Flt3L is a product of the Immunex Corporation (now Amgen).
B.J.W. and N.N. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Brenda J. Weigel, University of Minnesota Cancer Center, Department of Pediatrics, Division of Pediatric Hematology/Oncology and Blood & Marrow Transplant, MMC 366, 420 Delaware St SE, Minneapolis, MN 55455; e-mail: weige007{at}tc.umn.edu.
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Top
Abstract
Introduction
)
was maintained in RPMI-1640 culture medium supplemented with 1 mM
penicillin/streptomycin, 2 mM L-glutamine, and 10% fetal bovine serum
(FBS; HyClone, Ogden, UT). The cells were maintained at 37°C
and 5% CO2. C1498 cells grown in AIM-V serum-free media
(Life Technologies, Carlsbad, CA) were used to generate tumor cell
lysate by subjecting AML cells suspended in phosphate-buffered saline
(PBS) through 4 freeze/thaw cycles in liquid nitrogen and a 37°C
water bath. The cell viability was evaluated by trypan blue exclusion,
and no viable cells were present at the completion of 4 cycles. Lysates
were frozen at 
(TCR
B.