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Immunology |
Center for Genetic and Cellular Therapies, Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vaccination with tumor-derived antigenic mixtures obviates the need to know the identity of the relevant tumor antigens in each patient and will induce a polyclonal immune response against multiple targets on the tumor cell. It has been argued that the dominant antigens in the antitumor response are unique patient-specific tumor antigens, arisen by mutations incidental to the transformation process (6) . Thus, vaccination with antigens derived from autologous tumor, which would require the isolation of antigen from each cancer patient, may be superior to using a common source of antigens that could be obtained from allogeneic tumor cell lines. In animal models, vaccination of tumor-bearing mice using DCs loaded with autologous tumor-derived antigens in the form of tumor lysates (7 , 8) , peptides (9 , 10) , heat shock proteins (11) , or mRNA (12 , 13) was highly effective. The scope of vaccination with autologous tumor antigens is, however, limited by the availability of sufficient tumor tissue for antigen preparation, especially because it has been argued that continuous boosting will be required to maintain a protective antitumor immune response (14 , 15) .
Thus, many cancer patients may not benefit from current strategies of cancer vaccination because an effective tumor antigen associated with their cancer has not yet been identified or because sufficient tumor tissue cannot be obtained for antigen preparation.
We have shown previously that murine and human DCs3 transfected with mRNA can stimulate potent CTL responses in vitro and in vivo (12 , 16) , and treatment of tumor-bearing mice with dendritic cells (DC) transfected with tumor RNA led to a significant reduction in metastases (12) or survival benefit (13) . A key advantage of using antigen in the form of RNA is that RNA can be amplified from a few cells, and hence, sufficient, possibly unlimited, amounts of antigen can be generated from a limited amount of tumor tissue (17) . Because tumor cells can be isolated to a high degree of purity by microdissection from pathology slides, sufficient antigen could be generated from every cancer patient.
The purpose of this study was to determine whether RNA can be amplified from tumor cells with no loss of biological function. We show that murine DCs transfected with RNA amplified from the melanoma B16/F10.9 cell line are capable of priming CTL responses in mice and engender protective immunity in a postsurgical metastasis setting. Importantly, of added relevance to a clinical setting is the finding that human DCs transfected with RNA amplified from tumor cells microdissected from frozen tissue sections are capable of stimulating primary CTL responses in vitro.
This study provides the foundation for a potentially effective and broadly applicable treatment for cancer patients that does not require the characterization of the relevant antigenic profile in each patient and will not be limited by tumor tissue availability for antigen preparation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines
The B16/F10.9 melanoma clone of C57BL/6 origin is a highly
metastatic, poorly immunogenic, and a low class I-expressing cell line
(18)
. EL4 cells were established from a thymoma isolated
from C57BL/6 mice (ATCC# TIB39). Cells were maintained in DMEM
supplemented with 10% FCS, 25 mM HEPES, 2 mM
L-glutamine, and 1 mM sodium pyruvate. A cell
line derived by transfection of B16/F10.9 cells with a murine GM-CSF
cDNA plasmid, F10.9/GM, was used as a source of GM-CSF for generating
murine DCs. F10.9/GM cells were grown in RPMI 1640 supplemented with
5% FCS, 1 mM sodium pyruvate, 1 mM
nonessential amino acids, 100 IU/ml penicillin, 100 mg/ml streptomycin,
and 5 x 10-5 M
ß-mercaptoethanol and 10 mM HEPES. The human cell lines,
SW1463 (CEA+, HLA-A2 rectum adenocarcinoma) and
KLEB (CEA-, HLA-A2), were obtained from Dr.
Jeffrey Schlom (National Cancer Institute, Bethesda, MD). Human cell
lines were maintained in DMEM-F12 medium supplemented with 10% FCS, 25
mM HEPES, 2 mM L-glutamine, 1
µg/ml insulin, and 1 mM sodium pyruvate.
Preparation of DCs
Murine.
DCs were generated from bone marrow progenitors as described previously
(19)
. Bone marrow from tibias and femurs of C57BL/6 mice
were harvested, and the precursors were isolated. The precursors were
treated with ammonium chloride Tris buffer for 3 min at 37°C to
deplete the RBCs. GM-CSF-containing supernatant was harvested after
24 h from F10.9/GM cells and used at a final dilution of 1:10.
Cells were plated at 106/ml in GM-CSF-containing
medium and incubated at 37°C and 5% CO2. Three
days later, the floating cells (mostly granulocytes) were removed, and
the adherent cells were replenished with fresh GM-CSF-containing
medium. Four days later, the nonadherent cells were harvested (immature
day 7 DCs), washed, and replated at 106/ml in
GM-CSF-containing medium. After 2–3 days, the nonadherent cells were
harvested as DCs (mature day 9 or day 10 DCs), washed, and pulsed with
antigen.
Human.
DCs were generated from PBMCs, as described previously
(20)
. PBMCs were suspended at 6–7 x 106/ml in 30 ml of AIM V medium and transferred
to tissue culture flasks for adherence. After 1 h of adherence,
the nonadherent cells were removed, and 30 ml of AIM V medium with 800
units/ml GM-CSF and 500 units/ml IL-4 were added to the adherent cells.
After 7 days, the DCs were harvested and pulsed with antigen. DCs were
cryopreserved in 90% autologous plasma at 5 x 106/ml, and PBMCs to be used as responders were
cryopreserved at 5 x 107/ml.
Preparation of CEA and GFP RNA by in Vitro
Transcription
Cloning of pGEM4Z/CEA/A64.
Oligonucleotides containing 64 A-T bp, followed by an SpeI
restriction site, were placed between the EcoRI and
NarI sites of pGEM4Z (Promega Corp., Madison, WI) to create
the plasmid pGEM4Z/A64. A 2467-bp SmaI fragment isolated
from pGEM3Z-CEA (provided by Jeffrey Schlom) was ligated into the
SmaI site of pGEM4Z/A64 to create pGEM4Z/CEA/A64.
Linearization with SpeI, followed by in vitro
transcription with T7 RNA polymerase (Ambion mMessage mMachine kit,
Austin, TX), yields a transcript that contains 47 nucleotides of
vector-derived sequence, 85 nucleotides of CEA 5' untranslated region,
2106 nucleotides corresponding to the coding region of CEA, 265
nucleotides of CEA 3' untranslated region, 28 nucleotides of polylinker
sequence, 64 A nucleotides and 4 nucleotides from the SpeI
site.
Cloning of pGEM4Z/GFP/A64.
The 741-bp BamHI-NotI fragment containing the GFP
coding region was isolated from pEGFP-N1 (Clontech, Palo Alto, CA).
This fragment was cloned into the BamHI and NarI
sites of pGEM4Z (Promega), along with oligonucleotides containing a
NotI half-site, the pGEM4Z polylinker sequence from
BamHI to EcoRI, 64 A-T bp, an SpeI
site, and a NarI half-site to create pGEM4Z/GFP/A64.
Linearization with SpeI, followed by in vitro
transcription (Ambion mMessage mMachine kit), yields a transcript
containing 61 nucleotides of vector-derived sequence, the GFP coding
sequence, 24 nucleotides of polylinker sequence, 64 A residues, and 4
nucleotides from the SpeI site. RNA was transcribed in
vitro using T7 RNA polymerase and a cap analogue, as described
previously (12)
.
RT-PCR of Total Tumor RNA
RNA was isolated from tumor cells using the RNeasy kit
(Qiagen, Valencia, CA). One µg of total tumor RNA was reverse
transcribed in a final volume of 10 µl using SuperScript II reverse
transcriptase (Life Technologies, Inc., Gaithersburg, MD). First-strand
cDNA synthesis was primed with 10 pmol of a modified oligo-dT primer
[5'-AAGCAGTGGTATCAACGCAGAGTACT(30)VN-3'], where V is G, A, or C and N
is G, A, T, or C. The reaction was incubated at 42°C for 30 min prior
to the addition of 10 pmol of the T7 strand switch primer
(5'-CTAATACGACTCACTATAGGGCGGG-3'). The reaction was continued for 30
min and was stopped by placing it on ice. The cDNA was amplified by
placing 2 µl of the RT reaction into a 100-µl PCR reaction
containing 20 pmol of the following primers: T7 PCR
(5'-CCATCCTAATACGACTCACTATAGGGC-3') and 3' PCR (5'-
AAGCAGTGGTATCAACGCAGAGT-3'). Amplification was carried out under
standard conditions using Advantage cDNA Polymerase Mix (Clontech
Laboratories, Palo Alto, CA). Cycling conditions were as follows: an
initial 1 min denaturing step at 95°C, followed by cycling at 95°C
for 30 s, 65°C for 30 s, 68°C for 6 min, and a final
extension at 68°C for 7 min. The number of cycles performed was
dependent on the amount of RNA used in the RT reaction, and for 1 µg
of RNA, 18 cycles were typically performed. The amplified cDNA was
purified with a QIAquick PCR Purification Kit (Qiagen). The DNA was
ethanol precipitated, resuspended, and quantitated by UV
spectrophotometry.
In Vitro Transcription of Amplified cDNA
One µg of cDNA was placed in a standard in vitro
transcription reaction using a T7 mMessage mMachine Kit (Ambion). The
reaction was carried out at 37°C for 2–4 h, followed by the addition
of DNase I and incubation for 15 min. Ammonium acetate was added, and
RNA was isolated by phenol/chloroform extraction and isopropanol
precipitation. After centrifugation, the RNA pellet was resuspended in
RNase-free water, and LiCl precipitated to remove residual
unincorporated nucleotides. The pelleted RNA was resuspended in
RNase-free water, and the quantity and purity were determined by UV
spectrophotometry. An aliquot was electrophoresed on an
agarose/formaldehyde gel to determine the size range of the products.
Agarose/Formaldehyde Gel Electrophoresis and RNA Blotting
RNA was ethanol precipitated, dried, resuspended in gel loading
buffer containing formamide and formaldehyde, and loaded onto a 1%
agarose/formaldehyde gel. After electrophoresis, the RNA was stained
with ethidium bromide and then transferred to Hybond-N (Amersham
Pharmacia Biotech, Piscataway NJ) by electroblotting. The membrane was
prehybridized in Rapid-hyb buffer (Amersham Pharmacia Biotech) at
68°C for 30 min. A 537-bp fragment of murine ß-actin served as a
template for random-primed probe generation. The
32P-labeled probe was added to the membrane, and
hybridization was at 68°C for 2 h, followed by one wash in 2x
SSC, 0.1% SDS at room temperature for 20 min and two washes in 0.2x
SSC, 0.1% SDS at 68°C for 10 min each. The membrane was exposed to
film with an intensifying screen at -80°C.
Transfection of Murine and Human DCs with RNA
RNA, in 200 µl Opti-MEM, and the lipid DMRIE (Vical, San
Diego, CA), in 200 µl Opti-MEM, were mixed in polystyrene tubes at
room temperature for 5–10 min. The amount of CEA- or GFP-specific RNA
used was 1 µg, total tumor RNA was 10 µg, and amplified tumor RNA
was 5 µg, per 106 DCs. The RNA:lipid ratio was
1:3. The complex was added to DCs (5 x 106 cells/ml) in Opti-MEM (Life Technologies) and
incubated at 37°C for 20–30 min. DCs were washed and used as
stimulators.
CTL Priming in Mice
Bone marrow precursor-derived DCs were transfected with the
various RNA preparations, and naive, syngeneic mice were immunized i.v.
with 5 x 105 precursor-derived
DCs/mouse in 200 µl of PBS, two times at 14-day intervals.
Splenocytes were harvested 12–14 days after the final immunization and
depleted of RBCs with ammonium chloride Tris buffer. Splenocytes
(107) were cultured with 5 x 105 stimulator B16/F10.9 cells pretreated with
IFN-
and irradiated at 7500 rads in 5 ml of Iscove’s modified
Dulbecco’s medium with 10% FCS, 1 mM sodium
pyruvate, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 5 x 10-5 M
ß-mercaptoethanol/well in a six-well tissue culture plate. Cells were
cultured for 5 days at 37°C and 5% CO2.
Effectors were harvested on day 5 on Histopaque 1083 gradient prior to
use in a CTL assay.
Induction of Antigen-specific Primary Human CTL Responses in
Vitro
PBMCs were used as responder cells and resuspended in
complete RPMI [RPMI with 10% FCS, 2 mM
L-glutamine, 1 mM sodium pyruvate, 100 IU/ml
penicillin, 100 µg/ml streptomycin, and 5 x 10-5 M ß-mercaptoethanol] at
2 x 106 cells/ml. Cells were
cocultured with RNA-transfected DCs at a responder:stimulator
ratio of 10:1 in 10–20 ml of complete RPMI and 10 ng/ml IL-7. IL-2 was
added on day 3 at a concentration of 20 units/ml. Fresh medium was
added every 5 days. Viable cells were harvested on day 12, and
CD8+ T cells were isolated using CD8 microbeads,
per the manufacturer’s protocol (Miltenyi Biotech, Sunnyvale, CA). The
purity of CD8+ T cells was routinely 90% by
fluorescence-activated cell sorter analysis. The captured
CD8+ T cells were cultured in 10 ml of complete
RPMI 1640 and 20 units/ml of IL-2 at 37°C. Two days later, the
CD8+ T cell blasts were harvested and restimulated with RNA-transfected
DCs. CD8+ T cells were maintained at 5 x 105 cells/ml in complete RPMI, 10 ng/ml
IL-7, and 20 units/ml IL-2. CTL assays were done 5–6 days after
restimulation.
In Vitro Cytotoxicity Assay
Target cells (5–10 x 106)
were labeled with europium for 20 min at 4°C. Europium-labeled
targets (104
) and serial dilutions of effector
cells at varying E:T were incubated in 200 µl of complete RPMI 1640.
The plates were centrifuged at 500 x g for 3
min and incubated at 37°C for 4 h. Fifty µl of the supernatant
were harvested, and europium release was measured by time-resolved
fluorescence (21)
. Specific cytotoxic activity was
determined using the formula:
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A 3'-primer carrying a stretch of T residues was used to prime reverse
transcription. Reverse transcription is the limiting step in generating
full-length cDNAs and hence translatable RNAs. To enrich for cDNA
products that extended beyond the initiation codon of the mRNA (Fig. 1
,
ATG), reverse transcription was carried out with the RNaseH-minus
Moloney murine leukemia virus reverse transcriptase, which adds three
to four C residues to the end of the cDNA strand when the enzyme
reaches the 5' end of the RNA. This property can be used to anchor an
oligonucleotide, called "strand switch oligo," containing three G
residues, and permit the extension of the cDNA (22
, 23)
.
The sequence of the strand switch oligo used in this protocol
corresponds to the T7 phage promoter. The resulting cDNA is amplified
using the 3' primer (without the T residues) and the T7
promoter-containing oligo (without the G residues) as the 5'-primer in
a standard PCR reaction. The amplified cDNA product is used as the
template for RNA transcription using T7 RNA polymerase in the presence
of a 5'-cap analogue, as described previously (12)
. The
procedure, described in details in "Materials and Methods," is
completed in <24 h.
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. RNA was analyzed
before and after amplification by agarose gel electrophoresis. As
expected, staining with ethidium bromide (Fig. 2A)
shows that the predominant RNA species present before
amplification correspond to the two rRNA species, whereas the amplified
RNA migrates as a heterogeneous population corresponding in size to the
mRNA population in murine cells. To assess whether the amplified RNA
species correspond to full-length transcripts, the RNA displayed in
Fig. 2A
was blotted and hybridized with an actin-specific,
random-primed probe. In each case, a single prominent band was seen
that corresponds in size to the murine actin mRNA (Fig. 2B)
.
Judging from the band intensities, we estimate that the efficiency of
generating full-length actin during the amplification procedure was not
>3–5%. Note also that the actin-specific RNA species in the
amplified RNA preparation is slightly shorter than the actin RNA
species seen prior to amplification. This would be expected because 54
nucleotides are added to the ends of the amplified RNA species (30 A
residues + 23 residues corresponding to the 3' primer)
compared with 
200 A residues at the ends of cellular mRNA species.
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pretreated and irradiated B16/F10.9 cells, and tested for the
presence of B16/F10.9-specific CTLs using a standard cytotoxicity assay
(Fig. 3)
. As shown previously, irradiated B16/F10.9 were not capable of priming
a CTL response, reflecting the poor immunogenicity of this tumor cell
line (18)
. On the other hand, DCs transfected with tumor
RNA isolated from B16/F10.9 tumor cells or the amplified RNA products
stimulated a robust CTL response. The specificity of the CTL response
is shown by the fact that B16/F10.9 but not EL4 tumor cells were lysed
by the CTLs. The RT primer initially used in the amplification protocol
described in the legend to Fig. 1
contained a stretch of 30 T residues
to code for a 30-nucleotide long poly(A) tail in the amplified RNA.
Experiments in our laboratory have shown that extending the poly(A)
tail of in vitro-transcribed CEA RNA to 64 nucleotides
enhances the ability of the CEA RNA-transfected DCs to stimulate a CTL
response in vitro, presumably by increasing the stability of
the RNA in the
cell.4
As shown in Fig. 3
, CTL priming was improved by extending the poly(A)
tail of the amplified RNA from 30 to 64 residues. This difference was
seen reproducibly in three experiments using different RNA
preparations. Subsequent amplification protocols described below were
therefore modified to generate the longer poly(A) tail.
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should result in 30-fold
enrichment of mRNA sequences in the amplified product, reflecting the
abundance of rRNA in the starting material. Yet, as shown in Fig. 3
,
both pre- and postamplification RNA preparations induce a comparable
CTL response that cannot be explained by the fact that twice as much
preamplification RNA was used. The likely explanation for this
discrepancy, as indicated in Fig. 2
, is the low yield of full-length
translatable RNA species in the amplified product.
To determine whether priming of a CTL response shown in Fig. 3
correlates with a protective antitumor immune response, we tested
whether immunization of mice with amplified tumor RNA-transfected DCs
was capable of causing the regression of preexisting lung metastases.
Primary tumors were induced by implantation of B16/F10.9 tumor cells in
the footpad of mice. When the tumor reached a certain size (5.5–7.5 mm
diameter), they were surgically removed, and 2 days later, mice were
immunized with irradiated B16/F10.9 cells or with various
RNA-transfected DC preparations. Metastatic load was determined by
measuring lung weight and visual inspection (the latter is less
quantitative and more subjective). We have shown previously that in
this model, treatment of the tumor-bearing mice with B16/F10.9
RNA-transfected DCs exerts a pronounced antimetastatic response
(12)
. As shown in Fig. 4
, the lung weight of mice injected with PBS or treated with irradiated
B16/F10.9 cells quadruples to 0.8 g (average normal lung weight is
0.2 g), reflecting the extensive metastasis occurring in this
organ and the poor immunogenicity of B16/F10.9 tumor cells. On the
other hand, immunization with DCs transfected with either RNA isolated
directly from tumor cells or with amplified RNA exhibited a potent
antimetastatic response. The specificity of this response is shown by
the fact that DCs transfected with RNA from an unrelated tumor, EL4,
had no effect. In the pre- and postamplification B16/F10.9 treatment
groups, three and four animals, respectively, had normal lung weights
and were free of visible metastases, whereas in the control groups,
animals had multiple metastatic nodules that were too many to count.
The therapeutic response seen in the group treated with amplified RNA
was slightly superior to the group treated with nonamplified RNA. This
difference was seen in three experiments using three different
preparations of amplified RNA. The likely explanation, as discussed
above, is that the amplified RNA was enriched for mRNA species 30-fold
compared with the preamplification tumor RNA, which was offset in part
by the limited efficiency of the amplification process.
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and 4
show that RNA amplified
from tumor cells retains biological function. In the cancer patient,
the most readily available source of tumor is fixed tissue specimen.
Examination of pathology slides can accurately distinguish tumor from
benign tissue, and using microdissection techniques, tumor cells can be
isolated with a high degree of purity. In the following experiments, we
tested whether RNA isolated from tumor tissue microdissected from
pathology slides can be amplified without loss of function. Fig. 5A
shows an H&E-stained frozen section from a
CEA+ colorectal liver metastasis showing a
metastatic nodule surrounded by benign tissue. To isolate tumor cells,
normal tissue surrounding the cancerous nodule is first removed (Fig. 5B)
, and then the tumor cells are collected (Fig. 5C)
. Thirty-five nodules were isolated in a 3-h procedure
from two consecutive sections and pooled, and RNA was extracted. RNA
yield was 2.24 µg, obtained from 55,000 tumor cells. Gel analysis
under denaturing conditions and ethidium bromide staining confirmed
that the RNA was intact (data not shown).
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, DCs transfected with in vitro synthesized CEA RNA or with
RNA isolated from a CEA-positive cell line, but not from a CEA-negative
cell line, was capable of stimulating a CEA-specific CTL response. An
aliquot of the RNA isolated from the CEA-positive and the CEA-negative
cell lines was amplified as outlined in Fig. 1
. As shown in Fig. 6
, DCs
transfected with amplified RNA from the CEA-positive, but not from the
CEA-negative, cell line stimulated a CEA-specific CTL response. The
reduced magnitude of the CTL response generated with the amplified RNA
may represent either inefficient amplification or low level of CEA
expression in the cell line. Strikingly, a robust CTL response was also
generated using DCs transfected with RNA amplified from the
microdissected tumor cells. This experiment clearly shows that tumor
RNA can be amplified from tumor cells microdissected from tissue
sections without loss of immunological activity. The significance of
the differences seen in the magnitude of the CTL response stimulated
with the various RNA preparations is not clear and is under
investigation.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The amplification protocol outlined in Fig. 1
is simple and rapid; the
procedure involves well-characterized biochemical reactions carried out
in a test tube and is completed in <24 h. In the protocol used in this
study, RNA is amplified from 1 µg of total cellular RNA, which
corresponds to 40,000 cells. We have also successfully amplified RNA
from as little as 50 ng of tumor RNA (corresponding to 2000 cells).
A useful feature of this protocol is that the 5' primer used in the
amplification step encodes the phage T7 promoter. As a result, the
amplified cDNA library serves as the direct template for the in
vitro transcription of the RNA product. DCs transfected with
amplified cDNA instead of RNA would require the cloning of the cDNA
library into an expression plasmid (to provide a eukaryotic promoter),
which would be prohibitively complex to perform for each patient.
Gel analysis and ethidium bromide staining reveals that the size
distribution of the amplified RNA products corresponds to that of the
mRNA population in the cell (Fig. 2A)
. However, when a
specific RNA species was analyzed by blotting and hybridization, only a
fraction of the transcripts appear to be full length and hence
translatable (Fig. 2B
and data not shown). Yet, the
amplified RNA product appears to be equally or more effective than the
preamplified cellular RNA in stimulating CTL and protective antitumor
immunity (Figs. 3
, 4
, and 6)
. The likely explanation is that the
amplification protocol results in about 30-fold enrichment of the
relevant [poly(A)+] RNA population, which
compensates in part for the low frequency of the translatable RNA
species. Although current studies focus on improving the amplification
protocol, from a vaccination standpoint, the limited efficiency of the
amplification protocol is not a significant hindrance because
generating sufficient amounts of RNA is not a limiting factor.
The ability to amplify RNA from microdissected tissue illustrates the
power of the technology to provide an unlimited supply of antigen for
vaccination from a limited source of tumor tissue. Tumor cells are
often intertwined with benign tissue at the microscopic levels, as
illustrated in Fig. 5A
. Using microdissection techniques,
tumor cells can be isolated with a high degree of purity, preventing
the dilution of tumor antigens and reducing the potential of inducing
autoimmunity against self antigens from surrounding tissue. Concern
that vaccination with undefined antigenic mixtures from tumor cells may
also break tolerance to self antigens expressed in the tumor cells may
be addressed in the future by using subtractive hybridization
techniques (24
, 25)
, illustrating yet another potential
advantage of using tumor mRNA as antigen.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Department of Defense Grant
DAMD17-98-1-8304 (to E. G.). 
2 To whom requests for reprints should be
addressed, at Department of Surgery, Box 2601, Duke University Medical
Center, Durham, NC 27710. Phone: (919) 684-6465; Fax: (919) 681-7970;
E-mail: e.gilboa{at}cgct.duke.edu
3 The abbreviations used are: DC, dendritic cell;
GM-CSF, granulocyte/macrophage-colony stimulating factor; PBMC,
peripheral blood mononuclear cell; IL, interleukin; CEA,
carcinoembryonic antigen; GFP, green fluorescent protein; RT, reverse
transcriptase.
Received 7/19/99. Accepted 12/14/99.
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C. M. Coughlin, B. A. Vance, S. A. Grupp, and R. H. Vonderheide RNA-transfected CD40-activated B cells induce functional T-cell responses against viral and tumor antigen targets: implications for pediatric immunotherapy Blood, March 15, 2004; 103(6): 2046 - 2054. [Abstract] [Full Text] [PDF]
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M. R. Muller, G. Tsakou, F. Grunebach, S. M. Schmidt, and P. Brossart Induction of chronic lymphocytic leukemia (CLL)-specific CD4- and CD8-mediated T-cell responses using RNA-transfected dendritic cells Blood, March 1, 2004; 103(5): 1763 - 1769. [Abstract] [Full Text] [PDF]
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L. T. van den Broeke, E. Daschbach, E. K. Thomas, G. Andringa, and J. A. Berzofsky Dendritic Cell-Induced Activation of Adaptive and Innate Antitumor Immunity J. Immunol., December 1, 2003; 171(11): 5842 - 5852. [Abstract] [Full Text] [PDF]
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S. Nair, C. McLaughlin, A. Weizer, Z. Su, D. Boczkowski, J. Dannull, J. Vieweg, and E. Gilboa Injection of Immature Dendritic Cells into Adjuvant-Treated Skin Obviates the Need for Ex Vivo Maturation J. Immunol., December 1, 2003; 171(11): 6275 - 6282. [Abstract] [Full Text] [PDF]
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Y. Zhao, D. Boczkowski, S. K. Nair, and E. Gilboa Inhibition of invariant chain expression in dendritic cells presenting endogenous antigens stimulates CD4+ T-cell responses and tumor immunity Blood, December 1, 2003; 102(12): 4137 - 4142. [Abstract] [Full Text] [PDF]
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T. Satoh, T. Saika, S. Ebara, N. Kusaka, T. L. Timme, G. Yang, J. Wang, V. Mouraviev, G. Cao, E. M. A. Fattah, et al. Macrophages Transduced with an Adenoviral Vector Expressing Interleukin 12 Suppress Tumor Growth and Metastasis in a Preclinical Metastatic Prostate Cancer Model Cancer Res., November 15, 2003; 63(22): 7853 - 7860. [Abstract] [Full Text] [PDF]
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S. Santulli-Marotto, S. K. Nair, C. Rusconi, B. Sullenger, and E. Gilboa Multivalent RNA Aptamers That Inhibit CTLA-4 and Enhance Tumor Immunity Cancer Res., November 1, 2003; 63(21): 7483 - 7489. [Abstract] [Full Text] [PDF]
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 D. W. Mullins, S. L. Sheasley, R. M. Ream, T. N.J. Bullock, Y.-X. Fu, and V. H. Engelhard Route of Immunization with Peptide-pulsed Dendritic Cells Controls the Distribution of Memory and Effector T Cells in Lymphoid Tissues and Determines the Pattern of Regional Tumor Control J. Exp. Med., October 6, 2003; 198(7): 1023 - 1034. [Abstract] [Full Text] [PDF]
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M. R. Muller, F. Grunebach, A. Nencioni, and P. Brossart Transfection of Dendritic Cells with RNA Induces CD4- and CD8-Mediated T Cell Immunity Against Breast Carcinomas and Reveals the Immunodominance of Presented T Cell Epitopes J. Immunol., June 15, 2003; 170(12): 5892 - 5896. [Abstract] [Full Text] [PDF]
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 I. D. Davis, M. Jefford, P. Parente, and J. Cebon Rational approaches to human cancer immunotherapy J. Leukoc. Biol., January 1, 2003; 73(1): 3 - 29. [Abstract] [Full Text] [PDF]
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