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Immunology |
Departments of Surgery [M. H., K. H., M. T. L., H. T.], Molecular Genetics and Biochemistry [M. H., P. D. R., M. T. L., H. T.], and Cell Biology and Physiology [S. C. W.], School of Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213, and Department of Molecular Preventive Medicine, School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan [N. O., K. M.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.1% of administrated DCs). The DCs then form
clusters with adjacent lymphoid cells, which produce IFN-
(1500–3200pg/106cells/48 h) in response to tumor
stimulation. The number of the DCs migrating into lymph nodes were
greater when they were inoculated into the tumor rather than the skin.
Coculture of DCs and apoptotic tumor cells resulted in decreased
expression of CC chemokine receptor (CCR) 1 and increased CCR7
expression at mRNA level without alteration in other phenotypical
markers on DCs. Chemotaxis assay showed that CCR7 ligands, macrophage
inflammatory protein 3ß and secondary lymphoid-tissue chemokine
significantly (P < 0.05) induced the
migration of DCs when cocultured with apoptotic tumor cells. To
directly examine the involvement of CCR7 expression in DC migration, we
investigated the functions of DCs genetically modified to express high
levels of CCR7. CCR7 transduction promotes DC migration in response to
relevant ligands in vitro and in vivo.
These results suggest that the CCR7 expression of DCs is enhanced with
direct contact with apoptotic tumor cells and may have a critical role
for DC migrating to regional lymph nodes. The means to promote DC
delivery to tumor and to nodal sites represent novel targets for the
biological therapy of cancer. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent studies have demonstrated that chemokines play critical role in the migrating of DCs (11, 12, 13, 14, 15, 16, 17) . SLC is a new member of a subgroup of CC chemokines that initially seemed to be highly specific for lymphocytes and had no activity for monocytes (18, 19, 20, 21, 22) . SLC (ß chemokine 6-C-kine; also known as TCA4 or Exodus 2) is constitutively expressed in T-cell areas of lymph nodes, high endothelial venules, and mucosal lymphoid tissues. And this chemokine is an agonist for the MIP-3ß (ELC, also known as CKß-11) receptor, the CCR7 (also known as EBI-1 or BLR-2), which is a seven-transmembrane and G protein-coupled receptor. Recently, it has also been reported that mRNA expression of CCR1, CCR5, and CCR6 on DCs decreases progressively on their maturation, whereas CCR7 mRNA expression is up-regulated (11, 12, 13 , 16) .
In the current study, we evaluated whether that BM-DCs’ (cultured for 6 days with GM-CSF and IL-4) could effectively migrate from the tumor site to the draining lymph nodes and then induce Th-1 type response against tumor. It had been suggested that DCs failed to emigrate from tumor sites to draining lymph nodes, compromising the afferent aim of the immune response to tumor (7 , 23) . Surprisingly, we found that cocultivation of DCs with tumor cells induces CCR7 expression and suppresses CCR1 expression, and, furthermore, that CCR7 gene-transduced DCs migrate more efficiently to draining lymph. These results suggest that, even in the tumor setting, DCs migrate to draining lymph nodes through the dynamic changes in chemokine receptor expression (primarily CCR7) on DCs as occurs during other immune responses.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines.
MCA205 methylcholanthrene-induced fibrosarcoma was generously provided
by S. A. Rosenberg (National Cancer Institute, Bethesda, MD). This
syngeneic cell line for C57BL/6 mice was maintained in CM [RPMI
1640 supplemented with 10% heat-inactivated fetal bovine serum, 2
mM glutamine, 100 mg/ml streptomycin, 100 IU/ml penicillin,
and 5 x 10-5 M
2-melcaptethanol (all from Life Technologies, Inc., Grand
Island, NY)]. MCA205 was transduced with a retroviral vector
(DFG-GFP-Neo), which expresses both the GFP as well as the
neomycin-phosphotransferase (Neo) gene, and was then
selected with Geneticin (Life Technologies, Inc.) to obtain
MCA205-GFP-Neo. A retroviral vector, MFG-mCCR7 was also transfected to
MCA205 to obtain MCA205-CCR7. Primary culture of syngeneic fibroblasts
were obtained from the lung of C57BL/6 mice as follows. Animals were
killed, and the lungs were harvested. Small pieces of lung were minced
and stirred in a triple enzyme solution of collagenase IV,
hyaluronidase V, and DNase IV (Sigma, St. Louise, MO) for 3 h at
room temperature. After rinsing twice with HBSS (Life Technologies,
Inc.), cell suspensions were cultured in CM to obtain a primary culture
of fibroblasts.
Retroviral Vectors.
All of the retroviral vectors were created by subcloning the respective
fragments into MFG vectors as described previously
(24)
. The cDNAs of EGFP, Zeo, and CCR7 were obtained from
pEGFP-N1 (Clontech, Palo Alto, CA; Ref. 25
),
pcDNA3.1/Zeo(-) (Invitrogen, Carlsbad, CA), and pBS-SK(-)-mCCR7.
Retroviral supernatant was generated by transfecting these proviral
constructs into the BOSC23 packaging cell lines (26)
.
Culture of BM-DCs and Transduction with Retrovirus.
BM-DCs were obtained using methods described previously
(8, 9, 10)
. In brief, murine BM cells were harvested from the
femur and tibia of sacrificed mice. Contaminating erythrocytes were
lysed with 0.83 M NH4Cl buffer, and
lymphocytes were depleted with a cocktail of antibodies (RA3–3A1/6.1,
anti-B220; 2.43, anti-Lyt 2; GK1.5, anti-L3T4—all from American Type
Culture Collection, Rockville, MD) and rabbit complement (Accurate
Chemical and Scientific Corp., Westbury, NY) on day 0. These cells were
cultured overnight in CM to remove the adherent macrophages, and then
nonadherent cells were placed in fresh CM containing rmGM-CSF (1000
units/ml) and rmIL-4 (1000 units/ml; DC medium) on day 1 (Both
rmGM-CSF and rmIL-4 were kindly provided by Dr. Satwant Narula,
Schering-Plough Research Institute, Kenilworth, NJ). These
cells, cultured for 6 days in this condition, displayed a
characteristic morphology and surface phenotype compatible with a
constitutively immature population of DCs. Phenotypic analysis with
flow cytometry was performed on all of the preparations used in this
study to ensure the quality of the cell preparations used. DCs
primarily (60–90%) consisted of cells with moderately high expression
of CD11b, CD11c, CD86, CD80, CD54, and surface MHC class I and class II
molecules. For the retroviral transduction, 1 x 106 BM cells cultured in DC media for 1 day were
aliquoted to 14-ml round-bottomed tubes and suspended in 1 ml of the
retroviral supernatant with 8 µg/ml polybrane, 1000 units/ml of
GM-CSF and IL-4. These cells were centrifuged at 2500 x g at 30°C for 2 h (10
, 27
, 28)
. This
transduction process was repeated on days 3 and 4. The culture medium
of BOSC23 cells was used because these cells produced virus
supernatant at the highest titered (5 x 106 colony-forming units/ml). To examine
the transduction efficiency of murine BM-DCs, we used EGFP
gene as a marker and determined the efficiency of transduction by flow
cytometry. Transduction efficiency was in the range of
30–50% on day 5.
Fluorescence Labeling of Cells.
DCs were labeled with the red fluorescence marker PKH-26 (Sigma
Chemical Co., St. Louis, MO) according to the manufacturer’s protocol
(29
, 30) immediately prior to injection. In brief, DCs and
fibroblasts were incubated with 2 x 10-6 M PKH-26 at room temperature for
5 min, rinsed extensively with HBSS, examined for viability and number
using trypan blue exclusion, and injected into the animals. Viability
of DCs was more than 96% after labeling prior to injection.
Injection of DCs.
Mice were injected in the right flank i.d. with 1 x 105 MCA205 cells on day 0. On day 7, when the
tumor size reached approximately 10–20 mm2, HBSS
(0.1 ml) or 1 x 106 DCs, obtained
using the manner described above, were injected i.t.. Some animals were
injected in the right flank i.d. with these cells or with HBSS with no
preceding tumor inoculation.
Processing of Tissues for Immunohistochemistry.
Mice were killed at various time points (6, 24, 120 h, and 14
days) after injection, and tissues were harvested, fixed in 2%
paraformaldehyde and 30% sucrose after Zamboniis fixation for 12 h at 4°C, embedded in OCT compound, and immediately
frozen on dry ice. Serial 6-µm sections were made from these samples
using a cryostat and were examined by fluorescent microscopy (Olympus
BH-2, Tokyo, Japan). Some sections were stained with a mAb
specific against mouse CD11c or CD86 or with an isotype control
Ab (PharMingen, San Diego, CA) all of which were conjugated with FITC.
Evaluation of the results were performed in a blinded fashion.
Flow Cytometry.
For phenotypic analysis, DCs were stained with phycoerythrin- or
FITC-conjugated mAbs against murine cell-surface molecules (CD11b,
CD11c, CD80, CD86, CD54, Gr-1, H-2Kb, I-Ab, and appropriate isotype
controls, all from PharMingen) and examined with the FACScan (Becton
Dickinson, Sunnyvale, CA).
Coculture of DCs with Tumor Cells in Vitro.
Day-6 DCs (1 x 106) were
cocultured with 1 x 105 MCA205
tumor cells with or without UV exposure (31)
. Using a
UV-B (1590 µW/cm2 for 10–15 min),
2550% of the exposed MCA205 cells showed apoptotic change
determined by terminal deoxynucleotidyl transferase-mediated nick end
labeling assay as detected by APO-DIRECT (PharMingen) 1 day
after treatment (data not shown). In some conditions, DCs were
cocultured with UV-treated MCA205 cells, but direct cell-cell contact
between DCs and tumor cells was prevented using 0.4-µm-pore-size
transwell (Corning Coster, Cambridge, MA) inserted. On days 7 and 8,
cultured DCs were recovered and analyzed phenotypically using flow
cytometry. Total RNA was harvested from the remaining DCs and reverse
transcribed using standard methods to obtain cDNA. The resultant cDNA
was used as a template to detect mCCR1, mCCR7, and ß-actin expression
with PCR-specific primers.
Development of Quantitative Assay of DC Migration to Regional
Lymph Nodes Using Cytospin Evaluation.
We determined the number of migrating DCs found in regional lymph nodes
using a technique developed in our laboratory. Harvested lymph nodes
from the abdominal wall of the right flank were gently crushed and
suspended in PBS + 1% paraformaldehyde to obtain cell
suspension with 2 x 106 cells/ml.
This suspension (300 µl) was applied on a Cytofunnel chamber
(Shandon, Pittsburgh, PA), and centrifuged at 800 rpm for 3 min. The
slide was examined by immunofluorescence microscopy (Olympus BH-2 and
Olympus Provis AX-70, Tokyo, Japan), and the number of the
fluorescence-positive cells in the specimen was counted. The total
number of DCs migrating into the regional lymph nodes was determined as
the product of the number of cells per ml and the total volume (ml) of
the cell suspension. Evaluations of the results were performed in a
blinded fashion.
In Vitro Chemotaxis Assay.
An in vitro chemotaxis assay was performed as described
previously (11
, 13
, 32)
with minor modifications.
Recombinant chemokines (MIP-1
, MIP-3ß, and SLC; MIP-1 and
MIP-3ß were purchased from R&D System Inc., Minneapolis, MN) were
diluted with assay medium (the medium without FCS) to a final volume of
600 µl at appropriate concentrations and added to 24-well tissue
culture plates (Corning Coster, Cambridge, MA). Transwell culture
inserts (Corning Coster) with 6.5-mm diameter and 5.0-µm pore size
were inserted into each well, and DCs (3.5–5.0 x 105 cells per each well) were added to the top
chamber in assay medium at a final volume of 100 µl. After the plates
were incubated at 37°C in 5% CO2 for 4 h,
the cells in the bottom chamber were recovered, the migrating cells
were counted, and an aliquot was stained with anti-CD11c and
anti-CD86 mAbs to be analyzed by FACS,
In Vitro Cytokine Release Assay.
Lymphoid cells were obtained from the draining lymph nodes and spleen
(contaminating erythrocytes were lysed with 0.83 M
NH4Cl buffer) that were harvested from the mice
that had received i.t. injections with DCs 7 days earlier. These cells
(2 x 106) were cocultured with
2 x 105 irradiated (5000 rad)
MCA205 in 24-well plates for 48 h (9)
. The resultant
supernatant was collected and examined in an ELISA for mIFN- or
mIL-4 (PharMingen).
Analysis on mRNA Expression of Chemokines and Chemokine Receptors
Using RT-PCR.
Total RNA was isolated from various organs and DCs using RNAzol (Life
Technologies, Inc.) and was used for cDNA synthesis. The cDNAs were
used as templates for PCR (94°C for 1 min, 57°C for 1 min, and
74°C for 1 min, 26 cycles for mCCR1 and mCCR7, and 28 cycles for
mouse SLC) using specific primers for mCCR1 (forward:
5'-TCTAGTGTTCATCATTGGAGTGGTG; reverse: 5'-GACGCACGGCTTTGACCTTCTTCTC),
mCCR7 (forward: 5'-ACAGCGGCCTCCAGAAGAAGAGCGC; reverse:
5'-TGACGT-CATAGGCAATGTTGAGCTG), and mSLC (forward:
5'-CAACCACAACCATGGCTC; reverse: 5'-GGCGGGATCCTGGGCTAT). To ensure the
quality of the procedure, RT-PCR was performed on the samples using
specific primers for ß-actin.
Statistical Evaluation.
Statistical analysis was performed primarily using the unpaired
nonparametric study (Mann-Whitney’s U test). Some of the
analyses were performed by Student’s t test accompanied by
F test when applicable. Differences were considered
significant when the P value was less than 0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These cells were detectable within the
tumor for at least 5 days (data not shown). When labeled fibroblasts
were inoculated i.t., no cells were detected within the tumor at any
time point (data not shown). Within the regional (draining) lymph
nodes, labeled cells were observed only in animals treated with i.t.
inoculation of DCs (Fig. 1D)
but not fibroblasts (Fig. 1C)
. These fluorescence-positive cells formed clusters with
lymphoid cells within the lymph nodes (Fig. 2A)
. An immunohistochemical study using CD86 and CD11c
confirmed that these fluorescence-positive cells migrating into the
draining lymph nodes had phenotypic characteristics compatible with DCs
(data not shown).
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. Furthermore, more DCs were observed to form clusters
with the surrounding lymphoid cells (Fig. 2, A and C)
when compared with those of i.d. injected DCs (Fig. 2, B and C)
. Cluster formation is believed to be
characteristic of activated DCs. Lymphoid cells were harvested from the
draining lymph nodes and cocultured with MCA205 tumor cells for 48 h in vitro to analyze the expression of mIFN- and mIL-4
in the culture supernatant. Significant enhancement
(P < 0.05) of IFN- expression was
observed in samples obtained from the animals treated with i.t.
inoculation of DCs when compared with those treated with i.d.
inoculation of DCs and i.t. injection of HBSS (Fig. 2D)
. The
level of IL-4 was only marginally higher in animals after i.t. and i.d.
inoculation of DCs.
Coculture of DCs with Apoptotic MCA205 Cells Down-Regulates CCR1
Expression with Reciprocal Up-Regulation of CCR7 Expression on DCs.
We hypothesized that tumor cells might affect the expression of
chemokine receptors on DCs and modify the migratory capacity of DCs as
a result. To examine this hypothesis, we first cultured DCs admixed
with tumor cells that had been induced to undergo apoptosis using
UV-treatment in the schedule shown in Fig. 3
. CD11c and CD86 expression of DCs on day 7 and 8 were not altered after
coculture with normal or UV-irradiated tumor cells when compared with
that of DCs without coculture (Fig. 4)
. Expression of CD80 and surface MHC class II molecules was not altered
on day 7 and 8 either (data not shown), and their CD86 and CD80
expression was up-regulated at later time points (on day 9 and later;
data not shown). CCR1 and CCR7 expression of DCs was markedly
reciprocally down- and up-regulated, respectively, after coculture with
UV-treated MCA205 tumor cells (Fig. 5)
. DCs did not express much CCR7 message after coculture with UV-treated
tumor cells using an intervening transwell that prevents direct
DC-tumor interaction. Similarly, CCR1 expression of DCs did not
decrease after coculture with non-UV-treated MCA205 cells; and CCR7
expression of these DCs marginally decreased as compared with the DCs
that were cocultured with UV-treated MCA205 cells. DCs cocultured with
UV-treated, apoptotic MCA205 cells migrated more effectively in
response to CCR7 ligands in chemotaxis assay in vitro (Fig. 6)
, whereas DCs that were cocultured with non-UV-treated MCA205 had
decreased ability to migrate in response to SLC (data not shown). Thus,
DCs cocultured for 1 day with apoptotic tumor cells had significantly
enhanced ability to migrate in response to CCR7 ligands
(P < 0.05). Similar results were obtained
when another tumor cell line (B16) was used in place of MCA205 (data
not shown).
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. Whereas significant expression of SLC mRNA was detected in the
regional draining lymph nodes, no expression was detected in other
organs or DCs.
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. Furthermore, day-5 CCR7-DCs, which were retrovirally transduced with
the mCCR7 gene on days 24, expressed greater CCR7 message
than nontransduced DCs or Zeo-transduced DCs (Zeo-DCs). Endogenous CCR1
expression was unaffected after transduction with the different
retroviral vector. Flow cytometric analysis on various cell surface
molecules (I-Ab, H-2Kb,
CD80, CD86, CD54, and CD11c) revealed that CCR7 transduction had
no significant effect on DC phenotype (data not shown). An in
vitro chemotaxis assay showed that day-5 CCR7-DCs had better
migratory capability in response to SLC and MIP-3ß when compared with
nontransduced or Zeo-transduced DCs (P < 0.05; Fig. 9A
). Cells migrating to the bottom chambers were recovered and
analyzed by flow cytometry for CD11c and CD86 expression. No
significant differences were observed between these Zeo-DCs and
CCR7-DCs (data not shown). To examine the in vivo migratory
capability of these DCs, PKH26-labeled CCR7-gene- or
Zeo-gene-transduced DCs were inoculated i.t., and
fluorescence positive cells were counted in cells harvested from the
draining lymph nodes using cytospin evaluation as described in
"Materials and Methods." A significantly higher number of
fluorescence-positive cells were found in the lymph nodes of the
animals receiving CCR7-DCs than those receiving Zeo-DCs (Fig. 9B)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have also examined the characteristics of DCs migrating into the
draining lymph nodes using samples prepared for cytospin evaluation.
This method allowed us to perform a quantitative examination that was
not previously possible (33
, 34)
. Our studies showed that
a significantly (P < 0.05) greater number of
DCs inoculated i.t. migrate into the regional draining lymph nodes
within 24 h when compared with DCs inoculated i.d. Most (77%) of
the migrating cells formed clusters or interdigitated with surrounding
lymphoid cells, and these labeled DCs were detected for at least 5
days. This characteristic cluster formation between DCs and lymphoid
cells was as not frequently observed in samples prepared from
non-tumor-bearing animals treated with DCs inoculated i.d. Because
DC/T-cell cluster formation is a critical step for interaction, as
previously shown in ovalbumin model systems (34)
,
DCs that are inoculated i.t. seem to capture tumor-associated antigen
and present tumor-associated antigen peptides within the draining lymph
nodes. Lymphoid cells harvested from the draining lymph nodes produced
significantly higher quantities of IFN- in response to tumor cell
cocultivation in animals treated with i.t. inoculation of DCs in
vitro when compared with control groups. Furthermore, CTLs
in the draining lymph nodes and spleens of animals that were treated
with i.t. inoculation of DCs demonstrated potent tumor cell lysis that
can be inhibited with the incubation of H-2Kb
(MHC class I)-blocking Ab by 50–70% (data not shown). As
previously reported (31
, 35, 36, 37, 38, 39)
, there are many
spontaneous apoptotic cells within tumors in situ, and DCs
may acquire nominal tumor antigens and stimulate MHC class I-restricted
CTLs by phagocytosing these apoptotic tumor cells at the tumor site,
presenting them in both MHC class I and class II molecules.
Migratory capability of DCs is dictated by the change of responsiveness
of DCs to various chemokines during their development and maturation
(11, 12, 13)
. Immature DCs respond to MIP-3,
RANTES, and MIP-1 via chemokine receptors CCR1, -5, and -6,
whereas mature DCs respond to MIP-3ß/ELC (CKß-11) and SLC
via CCR7 (EBI-1, BLR-2) instead. Down-regulation of receptors for the
inflammatory chemokines (RANTES, MIP-1, MIP-1ß, and fMLP)
and up-regulation of receptors on mature DCs for chemokines (MIP-3ß
and SLC) that are expressed in secondary lymphoid organs allow DCs to
leave the sites of inflammation and antigen-uptake to migrate to
regional lymph nodes. In this study, we investigated the expression of
chemokine receptors on DCs at various stages of maturation in our tumor
model. We found that CCR1 mRNA expression of DCs cocultured with
UV-treated MCA205 cells is markedly down-regulated and CCR7 mRNA
expression is up-regulated. Interestingly, DCs that are
cocultured with UV-treated tumor cells separated by membrane inserts do
not express substantial CCR7 mRNA. These data suggest that it is
necessary for DCs to interact directly with apoptotic tumor cells to
induce changes in DC chemokine receptor usage. Moreover, these changes
in chemokine receptor expression were detectable even before observable
changes in expressions of CD86 and CD11c, both of which have been
previously used as markers to determine the maturity of DCs.
SLC is reported to be a novel CC chemokine with preferential expression in T-cell areas of lymph nodes, high endotherial venules, and mucosal lymphoid tissue (18 , 40) . Through the receptor CCR7, SLC and MIP-3ß can trigger rapid ß2 integrin activation in a major proportion of resting lymphocytes, inducing adhesion to ICAM-1 and promoting rapid lymphocyte arrest under conditions of physiological shear stress found in blood vessels. These chemokines can induce increased concentration of intracellular free Ca2+ and direct migration of mature DC via CCR7 (13 , 16 , 17) . In the present study, we have demonstrated that the DCs that are cocultured with apoptotic tumor cells express higher levels of CCR7 and enhance migratory capability in response to MIP-3ß and SLC. Both seem to be constitutively expressed from regional lymph nodes. These results suggest that CCR7 expression enhances migration of stimulated DCs from tumor sites into lymphatic vessels and lymph nodes, in which SLC is produced. Subsequently, MIP-3ß and SLC expression enhance local interaction between DCs and naive T cells in lymph nodes to induce an effective antitumor immune response (15 , 16 , 40 , 41) .
To directly examine the role of CCR7 in the migratory function of DCs, we examined the function of BM-DCs, which were retrovirally transduced with the mCCR7 gene. We demonstrated that CCR7 gene-transduced immature DCs (CCR7-DCs) have enhanced ability to migrate in response to SLC and MIP-3ß in vitro. Furthermore, CCR7-DCs that were inoculated i.t. migrated to the draining lymph nodes in vivo within 24 h after injection significantly better (P < 0.05) than control Zeo-DCs. Because CCR7 gene transduction does not seem to significantly alter the maturation markers of DCs, its expression in DCs seems to play an important role in promoting the migration of DCs from tumor sites to lymphoid organs in vivo. In our studies to date, we have not been able to demonstrate significantly enhanced antitumor effects in this setting (data not shown).
We have shown that immature DCs at tumor sites in contact with apoptotic tumor cells, acquire altered characteristics in their chemokine receptor usage and effectively migrate to the draining lymph nodes, which constitutively express the CCR7 ligand, SLC. Furthermore, we have shown direct evidence for the critical role of CCR7 expression required for the migration of DCs from the tumor site to draining lymph nodes with the induction of antitumor immune responses. This knowledge could be useful in developing improved strategies for DC-based therapy of cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grant PO1 CA59371 (to
M. T. L., P. D. R., and H. T.) and Grant 1PO1 CA73743-01 (to
M. T. L.) and by a Career Development Award of the American Society
of Clinical Oncology (to H. T.). 
2 Current affiliation: Department of Surgery and
Bioengineering, Institute of Medical Science, The University of Tokyo,
Japan.
3 To whom requests for reprints should be
addressed, at Department of Surgery, Institute of Medical Science, The
University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo, 108-8639,
Japan. Phone: 81-3-5449-5345; Fax: 81-3-5449-5444; E-mail: tahara{at}ims.u-tokyo.ac.jp
4 The abbreviations used are: DC, dendritic cell;
BM, bone marrow; BM-DC, BM-derived DC; GM-CSF, granulocyte macrophage
colony-stimulating factor; CCR, CC chemokine receptor; MIP,
macrophage-inflammatory protein; SLC, secondary lymphoid-tissue
chemokine; i.t., intratumoral; i.d., intradermal; rm, recombinant
murine; Th, T helper (cell); GFP, green fluorescence protein; EGFP,
enhanced GFP; IL, interleukin; CM, complete medium; Ab, antibody; mAb,
monoclonal Ab; RT-PCR, reverse transcription-PCR.
Received 10/ 7/99. Accepted 2/18/00.
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 K. Tschoep, T. C. Manning, H. Harlin, C. George, M. Johnson, and T. F. Gajewski Disparate functions of immature and mature human myeloid dendritic cells: implications for dendritic cell-based vaccines J. Leukoc. Biol., July 1, 2003; 74(1): 69 - 80. [Abstract] [Full Text] [PDF]
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J. J. Kobie, R. S. Wu, R. A. Kurt, S. Lou, M. K. Adelman, L. J. Whitesell, L. V. Ramanathapuram, C. L. Arteaga, and E. T. Akporiaye Transforming Growth Factor {beta} Inhibits the Antigen-Presenting Functions and Antitumor Activity of Dendritic Cell Vaccines Cancer Res., April 15, 2003; 63(8): 1860 - 1864. [Abstract] [Full Text] [PDF]
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I. J. M. de Vries, D. J. E. B. Krooshoop, N. M. Scharenborg, W. J. Lesterhuis, J. H. S. Diepstra, G. N. P. van Muijen, S. P. Strijk, T. J. Ruers, O. C. Boerman, W. J. G. Oyen, et al. Effective Migration of Antigen-pulsed Dendritic Cells to Lymph Nodes in Melanoma Patients Is Determined by Their Maturation State Cancer Res., January 1, 2003; 63(1): 12 - 17. [Abstract] [Full Text] [PDF]
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K. A. Tolba, W. J. Bowers, J. Muller, V. Housekneckt, R. E. Giuliano, H. J. Federoff, and J. D. Rosenblatt Herpes Simplex Virus (HSV) Amplicon-mediated Codelivery of Secondary Lymphoid Tissue Chemokine and CD40L Results in Augmented Antitumor Activity Cancer Res., November 15, 2002; 62(22): 6545 - 6551. [Abstract] [Full Text] [PDF]
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M. Nakamura, M. Iwahashi, M. Nakamori, K. Ueda, I. Matsuura, K. Noguchi, and H. Yamaue Dendritic Cells Genetically Engineered to Simultaneously Express Endogenous Tumor Antigen and Granulocyte Macrophage Colony-stimulating Factor Elicit Potent Therapeutic Antitumor Immunity Clin. Cancer Res., August 1, 2002; 8(8): 2742 - 2749. [Abstract] [Full Text] [PDF]
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E. Padovan, L. Terracciano, U. Certa, B. Jacobs, A. Reschner, M. Bolli, G. C. Spagnoli, E. C. Borden, and M. Heberer Interferon Stimulated Gene 15 Constitutively Produced by Melanoma Cells Induces E-Cadherin Expression on Human Dendritic Cells Cancer Res., June 1, 2002; 62(12): 3453 - 3458. [Abstract] [Full Text] [PDF]
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K. Mashino, N. Sadanaga, H. Yamaguchi, F. Tanaka, M. Ohta, K. Shibuta, H. Inoue, and M. Mori Expression of Chemokine Receptor CCR7 Is Associated with Lymph Node Metastasis of Gastric Carcinoma Cancer Res., May 1, 2002; 62(10): 2937 - 2941. [Abstract] [Full Text] [PDF]
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 G. M. del Hoyo, P. Martin, C. F. Arias, A. R. Marin, and C. Ardavin CD8alpha + dendritic cells originate from the CD8alpha - dendritic cell subset by a maturation process involving CD8alpha , DEC-205, and CD24 up-regulation Blood, February 1, 2002; 99(3): 999 - 1004. [Abstract] [Full Text] [PDF]
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T. Takayama, A. E. Morelli, N. Onai, M. Hirao, K. Matsushima, H. Tahara, and A. W. Thomson Mammalian and Viral IL-10 Enhance C-C Chemokine Receptor 5 but Down-Regulate C-C Chemokine Receptor 7 Expression by Myeloid Dendritic Cells: Impact on Chemotactic Responses and In Vivo Homing Ability J. Immunol., June 15, 2001; 166(12): 7136 - 7143. [Abstract] [Full Text] [PDF]
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H. Feng, Y. Zeng, L. Whitesell, and E. Katsanis Stressed apoptotic tumor cells express heat shock proteins and elicit tumor-specific immunity Blood, June 1, 2001; 97(11): 3505 - 3512. [Abstract] [Full Text] [PDF]
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 T. Kuhlmann, A. Bitsch, C. Stadelmann, H. Siebert, and W. Bruck Macrophages Are Eliminated from the Injured Peripheral Nerve via Local Apoptosis and Circulation to Regional Lymph Nodes and the Spleen J. Neurosci., May 15, 2001; 21(10): 3401 - 3408. [Abstract] [Full Text] [PDF]
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P. Hjelmström Lymphoid neogenesis: de novo formation of lymphoid tissue in chronic inflammation through expression of homing chemokines J. Leukoc. Biol., March 1, 2001; 69(3): 331 - 339. [Abstract] [Full Text]
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 M.-G. Roncarolo, M. K. Levings, and C. Traversari Differentiation of T Regulatory Cells by Immature Dendritic Cells J. Exp. Med., January 15, 2001; 193(2): f5 - f10. [Full Text] [PDF]
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F. Tanaka, W. Hashimoto, H. Okamura, P. D. Robbins, M. T. Lotze, and H. Tahara Rapid Generation of Potent and Tumor-specific Cytotoxic T Lymphocytes by Interleukin 18 Using Dendritic Cells and Natural Killer Cells Cancer Res., September 1, 2000; 60(17): 4838 - 4844. [Abstract] [Full Text]
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) or those forming clusters
(
) with surrounding lymphoid cells were enumerated as described in
"Materials and Methods." (Data are presented as mean ± SE.) D, significantly more DCs
(P < 0.05) formed clusters in the lymph
nodes when DCs were inoculated i.t. Lymphoid cells of the draining
lymph nodes were obtained from tumor-bearing mice 7 days after i.t.
inoculation of DCs (
). They were cocultured
with irradiated MCA205, and these supernatants were assessed for
IFN-
