|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 643-654
Eradication of Pre-Established Lymphoma Using Herpes Simplex
Virus Amplicon Vectors
By
Mahmood Kutubuddin,
Howard J. Federoff,
Pia M. Challita-Eid,
Marc Halterman,
Brian Day,
Meredith Atkinson,
Vicente Planelles, and
Joseph D. Rosenblatt
From the Department of Microbiology and Immunology, the Division of
Molecular Medicine and Gene Therapy, Department of Neurology,
University of Rochester Cancer Center, Department of Medicine,
University of Rochester, Rochester, NY.
 |
ABSTRACT |
Herpes simplex virus amplicon vectors expressing RANTES (HSVrantes)
and the T-cell costimulatory ligand B7.1 (HSVB7.1) were studied for
their ability to elicit a tumor-specific T-cell response in a murine
lymphoma model. HSVB7.1- and HSVrantes-transduced EL4 cells expressed
high levels of B7.1 and RANTES as analyzed by flow cytometry and
enzyme-linked immunosorbent assay, respectively. Inoculation of ex vivo HSVB7.1 transduced cells in syngeneic mice resulted in regression of both transduced cells and nontransduced cells
inoculated contralaterally. Direct intratumoral injection of HSVB7.1
and/or HSVrantes alone or in combination into established EL4
tumors led to complete tumor regression in injected tumors as well as
in nontransduced contralaterally implanted tumor, whereas control
tumors or tumors injected with HSVlac expressing  -galactosidase did
not regress. Maximal protection was achieved with combined injection of
HSVB7.1 and HSVrantes; mice showing tumor regression were resistant to
rechallenge with parental EL4 cells, and tumor cell-specific cytolytic
T-cell activity was observed in mice demonstrating regression. HSV
amplicon-mediated delivery of immune effector molecules may represent a
useful strategy for immunotherapy in the setting of pre-existing tumor.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
CONVENTIONAL THERAPIES for hematological
malignancies frequently fail to eradicate minimal residual disease.
Because malignancies of B- or T-cell origin possess antigen
presentation capabilities, the application of immunotherapy represents
an attractive strategy for elimination of minimal residual disease.
Peptide fragments derived from abnormal fusion proteins due to
chromosomal translocations, mutated oncogenes, and viral and
tumor-suppressor gene products may all serve as suitable immunologic
targets to T cells. Although such antigens may be presented by major
histocompatibility complex class I or II proteins (MHC-I/MHC-II),
hematologic tumors generally are not rejected. Failure of immune
rejection of these tumors may be partially attributed to a lack of
T-cell costimulation and consequent establishment of tumor-specific
tolerance by T cells. To generate protective immunity, neoplastic cells
need to deliver at least two signals to T cells reactive to tumor; first, an antigen-specific signal is delivered by antigenic peptide bound by MHC class I or II proteins to the antigen-specific T-cell receptor (TCR); second, a costimulus is communicated by members of the
B7 family (CD80 or B7.1, CD86 or B7.2, B7.x) via the T-cell counterreceptor CD28 and must be delivered within a short interval after the presentation of the first signal.1,2 Presentation of the first without the second signal may lead to the induction of
T-cell anergy3: such T cells lack the ability to mount a tumor-specific immune response and become tolerized.4 A
number of studies in humans and animals indicate that once established such tolerance cannot be reversed by costimulation with CD80
alone.5 However, combined CD80 stimulation and treatment
with cytokines can restore the ability of tolerized T cells to lyse
tumor cells.5-7 These data suggest that immunotherapy of
hematologic malignancies may require several interventions including
provision of a costimulatory signal, reversal of T-cell tolerance with
suitable cytokines,8 or the recruitment of nontolerized T
cells.
Chemokines are low molecular weight proteins that can attract T cells,
monocytes, dendritic cells, and other immune effector cells. RANTES, a
member of the C-C family of chemokines, is a potent chemoattractant of
monocytes as well as unstimulated CD4+, CD8+,
CD45RO+ memory T cells.9 RANTES also has the
ability to induce activation and proliferation of T cells when present
in high concentrations and may augment the effect of CD80
activation.10 RANTES expression has directly been shown to
generate a tumor-specific immune response in a mouse sarcoma
model.11
Tumor immunotherapy approaches involving gene transfer are based
primarily on the requirement of rapid high level expression of the
desired genes required to render a tumor immunogenic. A variety of
viral vectors, including retroviruses, adenovirus, vaccinia virus, and
others, have been used to enhance immunogenicity through delivery and
expression of cytokines, CD80, and other immune effector
molecules.7,12-15 Replication defective herpes simplex
virus (HSV) amplicon vectors offer several potential advantages in this
setting, including high transduction efficiency and expression levels
and the ability to package many copies of the transcriptional unit of
interest per virion.16-18 We have recently demonstrated the
successful use of HSV amplicon vectors expressing
granulocyte-macrophage colony-stimulating factor (GM-CSF) and
interleukin-2 (IL-2) to prevent hepatic metastases in a rat hepatoma
model.19
In this report, we evaluated the effectiveness of HSV amplicon vectors
to transduce CD80 and/or RANTES and elicit a protective immune
response to preestablished lymphoma. We demonstrate that in virtually
all animals transduction of CD80 and RANTES resulted in the eradication
of preestablished tumors, the generation of tumor-specific cytotoxic
T-cell (CTL) immunity, and immunologic memory.
| |
MATERIALS AND METHODS |
Cell Lines
EL4 cells were maintained in Iscove's Dulbecco modified Eagles Medium
(DMEM) with 10% fetal bovine serum (ID-10). EL4-B7.1 is an EL4 cell
line transduced with a Moloney murine leukemia virus-derived retroviral
vector encoding human CD80 cDNA.20 EL4-B7.1
cells were maintained in ID-10 containing G418 at 500 µg/mL
(GIBCO-BRL, Grand Island, NY). RR1 cells used for
packaging the HSVamplicon constructs are a BHK-derived cell line
engineered to stably express the HSV-IE3 gene.21 NIH 3T3
cells (ATCC, Rockville, MD) as well as the RR1 cell line
were maintained in DMEM with 10% (vol/vol) fetal bovine serum (FBS),
penicillin (100 µg/mL), streptomycin (100 µg/mL), and geneticin
(G418; 400 µg/mL; GIBCO-BRL). Murine splenocytes or enriched murine T
cells were maintained in RPMI-1640 medium with 10% FBS, penicillin,
and streptomycin, along with 5 × 10 5 mol/L
-mercaptoethanol (RP-10). YAC-1, a natural killer cell target cell
line, was maintained in RP-10 medium.
Construction of HSV Amplicon Plasmids
Construction of the HSV amplicon for human CD80.
The coding sequences for human CD80 or human RANTES were cloned into
the polylinker region of the pHSVPrPUC plasmid. Specifically, pBJ.huB7.1 plasmid (kindly provided by Dr Lewis Lanier, DNAX, Palo
Alto, CA) was digested with HindIII and filled in to generate a
blunt end and digested with Xba I. A
HindIII/Xba I fragment encoding the human CD80 (B7.1)
cDNA was gel-purified and used as insert in the ligation with the
vector. The HSV amplicon vector pHSVPrPUC was digested with
EcoRI and filled in with Klenow, followed by Xba I
digestion. The EcoRI/Xba I vector fragment was
gel-purified and ligated with the insert. Orientation of the coding
sequences of huB7.1 with respect to the HSV-1 IE4/5 promoter was
verified and the amplicon used in the generation of the HSVB7.1
amplicon virus.
Construction of the HSV amplicon for human RANTES.
The SK+pBS-RANTES plasmid (kindly provided by Dr Tom Schall,
ChemoCentryx, Mountain View, CA) was partially digested with Kpn I followed by digestion with Xba I. A Kpn
I/Xba I fragment encoding the human RANTES cDNA was
gel-purified and ligated to the HSV amplicon vector pHSVPrPUC plasmid
digested with Kpn I and Xba I. Orientation of the
coding sequences for huRANTES with respect to the HSV-1 IE4/5 promoter
was verified and the amplicon used in the generation of the HSVrantes
amplicon virus.
Virus Production and Titration
Amplicon DNA was packaged into HSV-1 particles by transfecting 5 µg of plasmid DNA into RR1 cells with lipofectamine as
recommended by the manufacturer (GIBCO-BRL). After incubation for 24 hours, the transfected monolayer was superinfected with the HSV strain 17, IE3 deletion mutant virus D30EBA22 at a multiplicity of infection (MOI) of 0.2. Once cytopathic changes were observed in the
infected monolayer, the cells were harvested, freeze-thawed, and
sonicated using a cup sonicator (Misonix, Inc,
Farmingdale, NY). Viral supernatants were clarified by centrifugation
at 5,000g for 10 minutes before repeat passage on RR1 cells.
This second viral passage was harvested as described above and
concentrated overnight by ultracentrifugation in a 25% sucrose
gradient as previously described.23 Viral pellets were
resuspended in phosphate-buffered saline (PBS; Ca2+ and
Mg2+ free) and stored at  80°C for future use.
Stocks were titered for helper virus by standard plaque assay methods.
Amplicon titers were determined as follows: NIH 3T3 cells were plated
in a 24-well plate at a density of 1 × 105 cells/well
and infected with the virus. Twenty-four hours after viral infection,
the monolayers were washed twice in PBS and either fixed with 4%
paraformaldehyde and stained by X-gal histochemistry (5 mmol/L
Potassium Ferricyanide; 5 mmol/L Potassium Ferrocyanide; 0.02% NP-40;
0.01% sodium deoxycholic acid; 2 mmol/L MgCl2; and 1 mg/mL Xgal dissolved in PBS) or harvested for total DNA using lysis
buffer (100 mmol/L NaCl, 10 mmol/L Tris, pH 8.0, 25 mmol/L EDTA, 0.5%
sodium dodecyl sulfate [SDS]) followed by phenol/chloroform extraction and ethanol precipitation. Polymerase chain reaction (PCR)
was performed on duplicate samples using primers corresponding to the
-lactamase gene present in the amplicon plasmid under the following
conditions: 94°C for 2 minutes; and then 20, 23, or 26 cycles of
94°C for 30 seconds and 58°C for 30 seconds, followed by
72°C for 7 minutes. PCR products from early and late cycles were
run on a 1% ethidium bromide gel, and the 450-bp band intensities were
assessed using the Fotodyne Foto/Eclipse system (Fotodyne, Inc,
Hartland, WI) and Collage Image Analysis Software. HSVB7.1 and
HSVrantes titers were estimated by comparison with HSVlac virus as
standards. Plaque forming unit (pfu/mL) and amplicon (bfu/mL) titers
obtained from these measurements were used to calculate amplicon titer
and thus standardize experimental viral delivery. Amplicon titer in the
different virus preparations ranged from 1 to 10 × 107 bfu/mL and the helper titers were in the range of 5 to
15 × 107 pfu/mL.
Flow Analysis for CD80 Expression
EL4 cells were infected in vitro either with HSVB7.1 or HSVlac amplicon
virus at an MOI of 1 pfu per cell. Specifically, 106 EL4
cells were adsorbed with the amplicon virus in a volume of 0.5 mL at
37°C, 5% CO2 for 4 hours. At the end of 4 hours, 0.5 mL of fresh ID-10 medium was added and incubation was continued for
another 12 hours. The infected cells were harvested after 16 hours and
106 cells in 0.1 mL of chilled PBS were stained with 1:10
diluted phycoerythrin-conjugated anti-B7.1 antibody (anti-CD80 PE;
Becton Dickinson, San Jose, CA) for 30 minutes at 4°C.
Uninfected EL4 cells (negative control) or EL4 stably expressing B7.1
(EL4-B7.1 as positive control) were also stained simultaneously with
the anti-CD80 PE antibody. The stained cells were analyzed by flow cytometry using an EPICS flow cytometry instrument.
In Vitro T-Cell Proliferation
T cells were enriched using a murine T-cell enrichment column (R&D
Systems, Minneapolis, MN). T cells (105) were
incubated in the presence of 5 × 104  -irradiated
stimulator cells. EL4 or CHO cells infected with HSV amplicon were used
as stimulator cells. Retrovirally transduced EL4-B7.1 (derived in the
laboratory) or CHO-B7.1 (kindly provided by Dr Peter
Linsley, Bristol Myers Squibb Pharmaceutical Research Institute, Seattle, WA) were used as positive controls for B7.1 expression and parental EL4 and CHO cells served as negative controls. Stimulator cells were irradiated with 7,500 rad using a
137Cesium-gamma source. Either anti-CD3 antibody (2C11)
used as 1:50 dilution of hybridoma cell supernatant or phorbol
myristate (10 ng/mL) with ionophore (0.1 ng/mL) was added and the cells
were cocultured for 3 days at 37°C. To assay for proliferative
responses, triplicate cultures were labeled for 16 hours with 1 µCi
3H-thymidine (NEN, Boston, MA; 2 Ci/mmol, 1 µCi/0.2 mL). Cells were harvested on glass fiber filters using a cell
harvester (Packard Instruments, Downers Grove, IL) and
incorporated 3H-radioactivity measured using a -counter
(Packard Instruments). Results are expressed as the mean (of triplicate
cultures) ± standard deviation. T-cell proliferation
index (normalized cpm) was determined as the ratio of
3H-thymidine incorporated in stimulated versus unstimulated
control cultures.
Enzyme-Linked Immunosorbent Assay (ELISA) for Analysis of RANTES
Production
EL4 cells were infected with HSVrantes or HSVlac amplicon at an MOI of
1. EL4 cells at 1 × 106 were adsorbed with the
amplicon virus in a volume of 0.5 mL at 37°C, 5% CO2
for 4 hours, and then 0.5 mL of fresh medium was added and incubation
was continued for another 20 hours. Cell culture supernatants were
harvested at the end of 24 hours and supernatants were tested for
RANTES in a sandwich ELISA using anti-RANTES antibody (R&D Systems) for
RANTES capture and biotinylated anti-RANTES (R&D Systems) for detection
followed by alkaline phosphatase-conjugated avidin.
Para-nitrophenyl phosphate was used as a substrate and absorbance at 405 nm was read in a Bio-Rad (Hercules, CA)
microplate ELISA reader. Serial twofold dilutions of standard
recombinant human-RANTES (R&D Systems) were run in parallel to
quantitate the amount of RANTES in the culture supernatant of infected
cells.
T-Cell Migration Assay
Primary murine T cells were purified using T-cell enrichment columns (R
& D Systems) from splenocytes of a normal C57BL6 adult mouse. T cells
(105) in X-Vivo 10 serum free medium (BioWhitaker Inc,
Walkersville, MD) were placed in the upper well of a transwell chamber
with a 3-µm pore size membrane (Costar Inc, Cambridge,
MA). Samples in lower wells contained either recombinant human RANTES
(R&D Systems) or HSVrantes-infected EL4 culture supernatant with known titer of RANTES. HSVlac-infected EL4 culture supernatant was used as a
negative control. All the samples were diluted in triplicate in X-Vivo
10 medium and an estimated concentration of 10, 1, or 0.1 ng/mL of
soluble RANTES was added to the lower wells. Control wells contained
the medium only and the background migration was monitored. The plates
were incubated at 37°C for 4 hours, and cells that migrated to the
lower wells were counted using a hemocytometer. The results are
represented as the mean ± SD of the number of migrated cells in the
lower wells assayed in triplicate.
Tumor Growth in Mice
Adult C57BL/6 (H-2b) female mice (8 weeks old) were
obtained from Charles River Laboratories (Wilmington, MA)
and maintained at the Animal Facility, University of Rochester Medical
Center (Rochester, NY). The mice were handled under an approved
laboratory animal handling and care protocol. Mice (6 per group) were
shaved on the dorsal side of the hind limb and inoculated
subcutaneously (SC) with 1 × 106 viable EL4
cells ex vivo infected at an estimated MOI of 1 with HSVB7.1,
HSVrantes, or HSVlac amplicon virus, or with uninfected EL4 cells. In
some experiments, 106 uninfected EL4 cells were inoculated
contralaterally at the same time on the other hind limb. Tumor growth
was measured every 2 to 3 days using a caliper and size reported in
millimeters diameter. Animals were killed when the tumor size exceeded
22 mm.
Intratumoral Delivery of HSV Amplicon
For intratumoral inoculation of the HSV amplicons, 106
viable EL4 cells were inoculated SC on the dorsal side of a shaved hind limb and the tumor was allowed to grow to a size of 5 to 6 mm (6 to 7 days). At this point, the mice were grouped and either HSVB7.1,
HSVrantes, HSVB7.1 + HSVrantes, or HSVlac amplicon virus diluted in PBS
to a concentration of 2 × 106 amplicon containing
virus particles in 50 µL was inoculated intratumorally (10 to 12 mice/group). Control animals with pre-established EL4 tumor received
only the diluent PBS. Flow cytometry analysis for B7.1 expression
performed 48 hours after intratumoral delivery of HSVB7.1 amplicon into
pre-established EL4 tumor on day 7 showed approximately 20%
B7.1-positive cells in several samples. The excised tumor cells were
stained immediately after the removal and dispersal of tumors (data not
shown). A second inoculation of the HSV amplicons was administered on
day 14, and the tumor growth was measured every 2 to 3 days. Tumors
were allowed to grow to a maximal size of 22 to 23 mm, at which point
the animals were killed. Mice from each group that showed no signs of
tumor growth at 1 month were selected for rechallenge with
parental EL4 cells. The secondary tumor growth after rechallenge was
monitored every 2 to 3 days.
In another experiment, 106 EL4 cells were inoculated
contralaterally at the same time on the both hind limbs. HSVB7.1 or
HSVrantes diluted in PBS to a concentration of 2 × 106 amplicon containing virus particles plus 2 × 106 HSVlac amplicon, 2 × 106 HSVB7.1 plus
2 × 106 HSVrantes, or 4 × 106
HSVlac amplicon virus diluted in PBS in 50 µL were injected into tumor on the right hind limb only on days 7 and 14 (10 mice/group). Tumor growth on both sides was measured every 2 to 3 days and size was
reported in millimeters diameter. Animals were killed when the tumor
size reached 22 to 23 mm.
CTL Assay
Spleens were harvested from C57BL/6 mice that had been inoculated with
EL4 cells and injected intratumorally with either HSVB7.1 or HSVrantes
or a combination of HSVB7.1 and HSVrantes. Control splenocytes were
obtained from mice that were inoculated intratumorally with HSVlac
virus or with PBS diluent alone. Splenocytes were prepared according to
standard procedures and red blood cells were lysed using AKC lysis
buffer. To obtain cytolytic T cells, splenocyte cell suspensions (2 × 106/mL in RP-10) were cultured together with
-irradiated (7,500 rad) EL4 cells (0.5 × 106
cells/mL) in a 25-cm2 flask at 5% CO2,
37°C for 6 days. These in vitro cocultured splenocytes were then
used as effector cells in the CTL assays. On the day of assay, EL4
target cells were washed with PBS and resuspended in RP10 medium (0.1 mL) at a concentration of 1.5 to 2 × 106 cells/mL and
Na51CrO4 (NEN; 100 µCi; stock concentration,
1 mCi/mL) added for 90 minutes at 37°C. These cells were washed 3 times with PBS and resuspended in 1 mL RP-10, and the viable cell count
was measured with a hemocytometer. 51Cr-labeled target
cells (l04 cells/0.1 mL) were added to the wells of a
V-shaped 96-well plate, and threefold serial dilutions of effector
cells were made in triplicate, resulting in final effector-target cell
ratios (E:T ratios) of 100:1, 33:1, 11:1, 3:1, and 1:1. Spontaneous
release of radioactivity from labeled target cells was measured by
culturing the target cells with medium alone in six wells. Total
release of radioactivity was determined by lysing the target cells with 2% Triton-X 100 detergent. Plates containing effector and target cells
were spun at 1,000 rpm for 2 minutes and incubated for 4 hours at 37°C, 5% CO2. The plates were then
centrifuged at 2,000 rpm for 4 minutes and half of the
culture supernatant (100 µL) was counted for 51Cr release
in a counter (Packard Instruments). Mean values are calculated for
the replicate wells and the results are expressed as the percentage of
specific lysis according to the formula: experimental counts spontaneous counts/total counts spontaneous counts × 100.
The mean spontaneous release for virus-infected and uninfected controls
averaged between 10% and 20% of the total counts.
Antibody Blocking
Monoclonal antibodies to murine CD4 (GK1.5), CD8 (3.155), or Thy-1
(30H-12) were used in the CTL assay to selectively block either
CD4+, CD8+, or Thy-1+ cells. The
effector cells for the antibody blocking assay were generated from
splenocytes of HSVB7.1 and HSVrantes amplicon inoculated mice with
known lytic activity as described above. These antibodies were used in
the 51Cr release assay as hybridoma culture supernatants
and were diluted 1:2 in a CTL assay. To test for the presence of
natural killer (NK) cell activity, the NK cell sensitive target Yac-1
cells (a lymphoma cell line) were used in the assay. Results of
triplicate cultures were expressed as the percentage of specific lysis
as described above. Average spontaneous release values ranged from 10%
to 20% of the total 51Cr incorporated.
| |
RESULTS |
Expression and Bioactivity of B7.1 in HSVB7.1 Amplicon-Infected Cells
EL4 T-lymphoma cells were transduced with the HSVB7.1 or HSVlac
amplicon virus (Fig 1). Infected cells were
harvested 24 hours later, immunostained for the expression of B7.1
using PE-conjugated anti-B7.1 antibody (anti-CD80 PE), and analyzed by
flow cytometry. Control uninfected EL4 cells or EL4 cells infected with
HSVlac were negative for B7.1 expression
(Fig 2A and B). In contrast, approximately
95% of EL4 cells infected at an estimated MOI of 1 stained positively
for B7.1 (Fig 2C). HSVB7.1 amplicon virus-infected EL4 cells showed
significantly higher levels of B7.1 expression than those seen with
retrovirally transduced EL4-B7.1 cells (data not shown). Expression of
B7.1 in HSVB7.1 infected cells was maintained for up to 60 hours
postinfection (data not shown).
View larger version (10K):
[in this window]
[in a new window]
| Fig 1.
HSV amplicon vectors. Coding sequences for human B7.1 or
human RANTES were cloned downstream of the HSV IE4/5 promoter in
pHSVPrPUC amplicon plasmid as indicated. HSVlac, which places the
Escherichia coli -galactosidase gene (lacZ) under
the transcriptional control of the IE 4/5 promoter, has been described
previously.19 Ampr denotes location of the
ampicillin resistance gene. OriS represents HSV-1
replication origin and Pac is the HSV-1 cleavage and packaging
sequence.
|
|
View larger version (17K):
[in this window]
[in a new window]
| Fig 2.
Expression of CD80 (B7.1) in HSVB7.1 amplicon-infected
EL4 cells. EL4 cells (A), EL4 cells transduced with HSVlac (B), or
HSVB7.1 (C) at an estimated MOI of 1 pfu/cell were stained with
PE-conjugated anti-CD80 antibody and analyzed by flow cytometry as
described in Materials and Methods.
|
|
The bioactivity of HSV vector-expressed B7.1 was studied in an in vitro
proliferation assay (Fig 3). Murine T cells
isolated from C57BL/6 mice were cocultured together with either
-irradiated EL4 or CHO cells that had been infected with either
HSVB7.1 or HSVlac as stimulator cells. Retrovirally transduced EL4-B7.1
or CHO-B7.1 cell lines were used as positive controls, whereas
untransduced EL4 or CHO cells served as negative controls. When
stimulated with anti-CD3 antibody (2C11) or a mixture of phorbol
myristate acetate (PMA) and ionophore to provide signal one, a
significant proliferative response was observed for T cells cocultured
with HSVB7.1- but not HSVlac-infected stimulator cells (Fig 3). The B7.1-dependent T-cell proliferative response observed with the HSVB7.1-infected EL4 cells was comparable to that seen with the retrovirally transduced control stimulator cells EL4-B7.1 or CHO-B7.1.
View larger version (29K):
[in this window]
[in a new window]
| Fig 3.
In vitro murine T-cell proliferation using HSVB7.1
amplicon-infected CHO or EL4 cells. Splenocytes from adult C57BL/6 mice
were harvested and T cells were enriched as described in Materials and
Methods. Purified T cells were cultured in triplicate wells with
HSVB7.1-infected CHO or EL4 cells. CHO-B7.1 or EL4-B7.1 cells stably
transduced with a retroviral vector expressing human B7.1 were used as
positive controls. HSVlac-infected or parental EL4 or CHO cells were
used as negative controls. Antimurine CD3 antibody (2C11) at a final
1:50 dilution or PMA (10 ng/mL) with Ionophore (0.1 ng/mL) was added as
indicated. After 72 hours, the cells were pulsed with
3H-thymidine and harvested and incorporated radioactivity
was measured as described in Materials and Methods. The results are
represented as the mean counts per minute (cpm) from triplicate
cultures ± standard deviation.
|
|
Expression and Bioactivity of RANTES from HSVrantes Amplicon-Infected
Cells
To monitor the production of soluble RANTES from HSVrantes-transduced
cells, EL4 cells were infected with HSVrantes or HSVlac at an MOI of
0.5. Conditioned media from culture supernatants were collected 24 hours later and analyzed for RANTES by quantitative ELISA. In
uninfected EL4 cells or cells transduced with HSVlac, no detectable
RANTES secretion was observed in culture supernatants. Cells infected
with HSVrantes at an MOI of 0.5 produced 3.1 ng of RANTES/mL/24
hours/106 cells. The observed levels of RANTES were higher
than those measured in pooled G418 selected retrovirally transduced
EL4-RANTES cells that secreted RANTES at a concentration of 1.45 ng/mL/24 hours/106 cells.
To test for the bioactivity of soluble RANTES from HSVrantes-infected
EL4 cells, culture supernatant from HSVrantes-transduced cells was
tested in a murine T-cell migration assay using a transwell chamber.
Culture supernatant from HSVlac-transduced cells was used as a negative
control. Recombinant human RANTES was used as a positive control.
Control migration of murine T cells was also tested in response to
diluent medium alone. Observed migration in response to
HSVrantes-infected EL4 culture supernatant was significantly higher
than that seen with the HSVlac or the control medium and comparable to
that observed with corresponding dilutions of recombinant RANTES
(Table 1). This demonstrated that soluble RANTES secreted by the HSVrantes-infected EL4 cells is capable of
eliciting a chemotactic response in murine T cells.
Growth of HSV Amplicon-Transduced EL4 Cells in Mice
In preliminary experiments, the growth of ex vivo HSV amplicon-infected
EL4 cells was measured in adult C57BL6 mice. After transduction with
the HSV amplicon virus at an MOI of 1, 106 viable infected
cells were inoculated SC in C57BL6 mice and tumor size was measured
every 2 to 3 days. The results are summarized in
Table 2. On day 20, complete regression of
tumor was noted in 3 of 6 mice inoculated with HSVB7.1-infected EL4
cells, whereas 2 of 6 mice inoculated with HSVrantes-infected EL4 cells
showed initial tumor growth followed by complete regression. When EL4 cells were infected with both HSVB7.1 and HSVrantes, 5 of 6 mice showed
complete regression after initial tumor growth. Although some mice
demonstrated initial tumor growth before regression, regression
generally began within 7 to 10 days of tumor inoculation, suggesting a
gradually increasing antitumor response. Contralateral nontransduced
EL4 cells showed a delay in regression of 2 to 3 days relative to
transduced cells (Fig 4A).
Control EL4 cells or EL4 cells infected with the HSVlac vector grew in
100% of the mice (6 of 6), whereas stably transduced EL4-B7.1 cells
showed no evidence of tumor growth in 6 of 6 mice by day 20 (data not shown). These results suggest that HSVB7.1 or HSVrantes
amplicon-infected cells may have been rejected due to a tumor-specific
immune response.
View larger version (15K):
[in this window]
[in a new window]
View larger version (16K):
[in this window]
[in a new window]
| Fig 4.
Growth of HSVB7.1 or HSVlac amplicon-transduced EL4 cells
and parental EL4 cells injected contralaterally in C57BL/6 mice. EL4
cells were infected ex vivo at an estimated MOI of 1 with either
HSVB7.1 (A; mice no. 1 through 5) or HSVlac virus (B; mice no. 6 through 10). Viable transduced EL4 cells (106) were
implanted SC on one side of the hind limb of the C57BL/6 mice, and
parental (nontransduced) EL4 cells (106 cells/per mouse)
were implanted on the contralateral hind limb. Tumor diameter was
measured and expressed in millimeters. Tumor size in individual animals
is shown.
|
|
We next evaluated whether inoculation of HSV vector-transduced cells
would inhibit growth of concurrent contralaterally inoculated parental
nontransduced EL4 cells. In 3 of 5 mice, regression of ex vivo
HSVB7.1-infected EL4 tumor was concordant with regression of a
contralaterally implanted EL4 cell (Fig 4A). Both HSVlac-infected EL4
cells and contralateral parental EL4 cells developed into tumor in 5 of
5 animals studied (Fig 4B). These data suggest that systemic
tumor-specific immunity to parental EL4 cells had developed in a subset
of mice inoculated with HSVB7.1-transduced EL4 cells.
Intratumoral Delivery of the HSV Amplicon Vectors
To test the effect of HSVB7.1 and HSVrantes on growth of established
tumors in mice, 106 viable EL4 cells were inoculated SC in
the hind limb of mice, and tumors were allowed to grow to a diameter of
5 to 6 mm (6 to 7 days). On days 7 and 14, HSV amplicons were delivered
intratumorally (10 to 12 mice/group) and tumor size was measured every
2 to 3 days (Fig 5). In three combined
experiments, complete tumor regression was observed in 17 of 26 (65%)
mice injected with HSVB7.1 vector alone, in 11 of 22 (50%) mice
injected with HSVrantes, and in 23 of 26 (88%) mice injected with the
combination of HSVB7.1 and HSVrantes (Fig 5). Although additional tumor
growth was initially observed, rejection of both HSV amplicon-injected
and noninjected tumor generally occurred within 7 to 14 days. A modest
lag was observed in rejection of contralateral tumor in some mice,
suggesting that gradual amplification of the systemic response was
necessary. To determine whether regression of tumor correlated with the
development of T-cell memory, mice manifesting complete tumor
regression after 1 month were rechallenged with 106 viable
EL4 cells in the hind limb contralateral to the primary inoculation.
All mice rechallenged with EL4 cells showed no evidence of tumor growth
at 1 month after rechallenge (Fig 5), indicating that systemic immunity
had been established by the antecedent direct intratumoral delivery of
HSVB7.1 and/or HSVrantes into pre-established tumors. Some mice
were observed for 2 to 3 months, and no relapses were observed (data
not shown).
View larger version (12K):
[in this window]
[in a new window]
View larger version (20K):
[in this window]
[in a new window]
View larger version (14K):
[in this window]
[in a new window]
| Fig 5.
Tumor incidence in mice after inoculation of HSV amplicon
vector into pre-established EL4 tumor. (A) Viable EL4 cells
(106) were implanted SC on one side of the hind limb of the
C57BL/6 mice (8 weeks old). Tumors were allowed to develop to a size of
5 to 6 mm in diameter. HSV amplicon virus (2 × 106
amplicon-containing particles) was inoculated on day 7 and again on day
14, and tumor growth was monitored every 3 days. The graph represents
the percentage of tumor-bearing mice over time. ( ) HSV-B7.1; ( )
HSVrantes; ( ) HSV-B7.1 + HSVrantes; ( ) HSVlac. The number of
animals used in each group is shown in (B). Results of three separate
experiments are pooled. Mice in which primary tumor had regressed were
selected for rechallenge with 106 viable EL4 cells and
tumor growth was observed for another month. Mice were killed when the
tumor diameter exceeded 22 mm. (C) Statistical analysis was performed
using Fisher's exact test comparing all four arms to each other.
|
|
In a separate experiment, we tested whether the delivery of HSV
amplicon into pre-established tumor resulted in induction of systemic
immunity against a contralateral tumor inoculated at the same time on
the left hind limb. Viable EL4 cells (106) were inoculated
SC bilaterally on day 0, and HSV amplicon vectors were injected on days
7 and 14 to the tumor established on the right hind limb (10 mice/group; Fig 6). Tumor growth on both
hind limbs was measured every 3 to 4 days. Complete tumor regression was seen in 5 of 10 mice (50%, P = .0325) inoculated with
HSV-B7.1, 5 of 10 (50%, P = .0325) with HSVrantes, and 8 of 10 (80% P = .0007) in combined HSV-B7.1 and HSVrantes-treated
animals. The P value is calculated relative to the HSVlac
control group. In animals treated with HSVB7.1 alone (5 of 5, P = .0325) or with the combination of HSVB7.1 and HSVrantes (8 of 8, P = .0007), regression of the contralateral untreated tumor was
consistently observed along with the treated tumor. In mice injected
with the HSVrantes vector alone, of the 5 mice in which the injected
tumor regressed, only 4 of 5 contralateral tumors regressed completely (P = .1734). The P value is calculated relative to the
untreated tumor in HSVlac control group. In one of 5 HSVrantes-treated
animals, the contralateral tumor grew at a reduced rate relative to
control untreated animals. In the HSVlac-treated animals, 10 of 10 animals demonstrated tumor growth on both sides. We noted that
HSVlac-treated tumors grew at a slightly reduced rate compared with
control untreated tumors. Using a two-sided log rank test, a
statistically significant effect was seen for both the treated and
untreated contralateral tumor after treatment with HSVB7.1
and/or HSVrantes compared with the control HSVlac-injected
group. Although an increased number of animals were tumor free after
the combined use of HSVB7.1 and HSVrantes, there was no statistical
significance compared with treatment with HSVB7.1 (P = .2823)
or HSVrantes alone (P = .3223). This demonstrates that systemic
immunity generated as a result of intratumoral HSV amplicon injection
could prevent development of contralateral noninjected tumor.
View larger version (13K):
[in this window]
[in a new window]
| Fig 6.
Tumor incidence in mice inoculated with HSV amplicon into
pre-established EL4 tumor and growth of parental EL4 cells
contralaterally. Viable EL4 cells (106) were implanted SC
on both hind limbs of 8-week-old C57BL6 mice. Tumors were allowed to
develop to a size of 5 to 6 mm diameter. HSV amplicon virus (2 × 106 amplicon-containing virus particles) was injected into
the right tumor on days 7 and 14, and growth of the HSV
amplicon-treated () and untreated EL4 tumor ( ) was monitored
every 3 days. Growth of HSVlac-treated tumor () or contralateral
untreated EL4 tumor () are also shown. Each experimental group
consisted of 10 mice. The graph represents the percentage of
tumor-bearing mice over time. Mice were killed when the tumor diameter
exceeded 22 mm.
|
|
CTL Response
To examine the induction of CTL responses in mice transduced
intratumorally with the HSV amplicon vectors, splenocytes from these
mice or from mice rechallenged with EL4 cells after primary tumor
regression were cocultured in vitro along with irradiated stimulator
EL4 cells for 6 to 7 days. Such in vitro boosted splenocytes were used
at different effector to target ratios and 51Cr release
from a fixed number of labeled EL4 cells counted as a measure of CTL
activity. Significant CTL activity was seen in splenocytes from mice
receiving HSVB7.1 or HSVrantes alone or in combination
(Fig 7A). CTL responses were only seen in
mice in which EL4 tumor regressed after direct delivery of the HSVB7.1 and/or HSVrantes amplicons into pre-established tumor. Little or no lytic activity was seen with splenocytes from mice treated with
the HSVlac amplicon. Consistent with the absence of secondary tumor
growth upon rechallenge, CTL activity was also observed in mice which
had been rechallenged with the parental EL4 cells (data not shown).
View larger version (18K):
[in this window]
[in a new window]
View larger version (12K):
[in this window]
[in a new window]
| Fig 7.
(A) CTL activity in mice splenocytes after intratumoral
inoculation with HSV amplicon vector. EL4 cells were inoculated SC in
mice and tumors allowed to grow to 5 to 6 mm in diameter over 6 to 7 days. On days 7 and 14, HSV amplicon virus was injected as indicated
directly into the tumors. Spleens were harvested 1 month later and
splenocytes were cultured in vitro in the presence of irradiated EL4
cells. After 6 days in culture, CTL activity was measured by release of
51Cr from labeled EL4 cells. CTL activity of splenocytes
harvested from individual mice intratumorally injected with HSVB7.1
(, ,  ) HSVrantes (,  , ) HSVB7.1 and HSVrantes (,
 ,  ), or HSVlac (*, ×) are shown. Data are expressed as the
percentage of specific lysis versus E:T ratio. (B) Antibody blocking of
the CTL activity with anti-CD4, anti-CD8, anti-Thy-1, or anti-T-cell
cocktail (anti-CD4, anti-CD8, and anti-Thy-1) antibodies. Splenocytes
with known CTL activity (harvested from HSV-B7.1+ HSVrantes
inoculated mouse no. 26 [A]) were used in an antibody blocking assay.
No antibody (), anti-CD4 (GK1.5, ), anti-CD8 (3.155, ), or
anti-T-cell antibody cocktail (anti-CD4, anti-CD8, and anti-Thy-1,
) were added to the CTL assay at the indicated effector:target
ratios as described in Materials and Methods. In a separate experiment,
Yac-1 cells were used as target cells (*) for measuring NK activity.
Data are represented as the percentage of specific lysis versus E:T
ratio.
|
|
Monoclonal antibodies recognizing murine CD4, CD8, or Thy-1 markers
were used to further characterize the CTLs in the effector population.
CTL assays were performed using effector cells from mouse no. 26 (HSVB7.1 and HSVrantes-treated with complete tumor regression) in the
presence of antibodies to either CD4, CD8, or Thy-1. Lysis was markedly
inhibited in the presence of either an anti-T-cell monoclonal antibody
cocktail (CD4,CD8, and Thy-1) or anti-CD8 antibody, but not by anti-CD4
antibody (Fig 7B). To test whether NK cell lysis activity was present,
51Cr-labeled Yac-1 cells were used as alternative targets
in a CTL assay. Low levels (~20%) of NK activity were detected at
the highest effector:target ratio (100:1) but not at lower
effector:target ratios (Fig 7B). Therefore, we conclude that the
predominant effector population consisted of CD8+ CTLs.
| |
DISCUSSION |
Eradication of minimal residual disease remains a central problem in
the treatment of hematologic malignancies. In this report, we describe
complete eradication of pre-established lymphoid tumors in
immunocompetent mice using HSV amplicon-mediated delivery of the T-cell
costimulatory ligand CD80 and the chemokine RANTES.
With rare exception, expression of CD80 and/or CD86 is low to
absent on tumors of both hematopoietic and nonhematopoietic origin.24 Expression of either CD80 or CD86 through gene
transfer has therefore been used to generate tumor-specific
immunity.25-28 In several studies, transfection of either
CD80 or CD86 into immunogenic tumors resulted in the generation and
establishment of antitumor immunity that offered long-term protection
against challenge with B7-negative parental tumor
cells.25-28 CD80 expression using retroviral vectors has
been shown to confer immune protection and memory against EL4 cells as
well as other tumors, including myelocytic leukemia and
adenocarcinoma.13,14,25,26 Most of these studies involved
primary immunization with CD80 transduced cells followed by rechallenge
with the parental tumor cells. In a study with a bcr/abl-transformed
myeloid leukemic cell line, a single exposure of mice to CD80
transduced leukemic cells conferred protective immunity.29
In addition, a modest challenge using a lethal dose of parental
leukemic cells could be rejected if mice were repeatedly immunized with
ex vivo CD80-transduced cells starting 1 day after inoculation of the
tumor challenge. However, if hyperimmunization was delayed greater than
3 days, protective effects were not seen. A similar requirement for
repeated inoculation was demonstrated for M1 leukemic
cells.30 In another study using both CD80- and IL-2-transduced cells, prolonged survival and delayed growth of NC
tumor was observed after implantation of transduced cells on days 7, 18, and 33 posttumor implantation.7 These results suggested that vigorous and repeated immunization with cells expressing CD80
alone or with IL-2 may be required for an immune response to parental
tumor. In the present study, we demonstrate that intratumoral inoculation with HSVrantes and/or HSV B7.1 into
well-established tumors could produce sustained antitumor immunity. Our
data suggest that direct viral inoculation in vivo into preexisting
tumor may be similar to hyperimmunization and that the levels of
CD80/chemokine expression after direct HSV amplicon vector injection
were adequate to confer protection.
Although costimulation of T cells through the CD80-CD28 pathway has
been shown to induce the synthesis of the chemokine macrophage inflammatory protein 1 (MIP1), other C-C
chemokines, such as MIP1, lymphotactin, or RANTES, are not
induced.31 Therefore, combined expression of CD80 and
RANTES may potentiate different pathways than those induced by CD80
alone. RANTES may also enhance response to CD80 costimulation and may
directly elicit an antitumor response at high concentrations in a
murine sarcoma model.11 The in vitro levels of RANTES
observed in this study were similar to those seen with other molecules,
such as interferon- (IFN-), GM-CSF, or nerve growth
factor (NGF), expressed through the use of HSV
amplicons.19,23,32 It is possible that higher
concentrations of RANTES could be achieved in vivo if higher virion
doses were administered.
Although either HSVB7.1 or HSVrantes conferred partial protection,
combined use of both vectors resulted in eradication of about 88% of
established tumors. It has been reported that expression of IL-2 in
combination with CD80 expression may be effective in reversing
tumor-induced tolerance in follicular lymphomas.6 Examining
a murine B-cell lymphoblastic leukemia model, Dilloo et
al33 reported that CD80 induction using CD40 ligand or IL-2 treatment alone showed partial protection from tumor growth, whereas enhanced response was seen using a combination of CD40 ligand and IL-2.
In the NC adenocarcinoma model, neither CD80 nor IL-2 alone had any
significant effect on NC tumorigenicity. However, combined expression
of CD80 and IL-2 substantially decreased NC tumorigenicity in
mice.7 We are currently testing whether combined HSV
amplicon delivery of cytokines such as IL-2, IFN-, or other chemokines may further enhance the observed protective response in the
EL4 T-lymphoma model. Other investigators have demonstrated that
recruitment using the chemokine lymphotactin, coupled with IL-2
stimulation, may be superior to the use of either alone in eliciting an
antitumor response.34 Although RANTES is known to
predominantly attract memory T cells, attraction of naive T cells or
other T-cell subpopulations using other chemokines such as the recently
characterized DC-CK135 or lymphotactin34 may further potentiate an antileukemic response. Nevertheless, these results suggest that expression of RANTES alone or in combination with
CD80 is a useful strategy for eliciting an immune response to
pre-established lymphoma.
We were successfully able to eradicate pre-established EL4 tumors in
mice by intratumoral inoculation of HSVB7.1 and/or HSVrantes amplicon virus. If these two are combined and inoculated, an increased number of animals reject the tumor. We have also demonstrated that
systemic tumor-specific immunity can be established, because mice that
reject the HSV amplicon-injected EL4 tumor also rejected a
contralateral challenge with parental tumor. A short delay in onset of
tumor regression in contralateral uninjected tumors was seen,
suggesting that 10 to 14 days were necessary for adequate amplification
of a systemic antitumor response to facilitate rejection. We
consistently observed that mice that had rejected the tumor maintain a
tumor-specific memory T-cell response, because 100% of such mice were
protected upon rechallenge with parental tumor cells. Tumor regression
also correlated with the development of tumor cell-specific CTL
responses in mice. Expression of CD80 in EL4 cells has been known to be
immunogenic in C57BL/6 mice and this immunity was shown to be mediated
by CD8+ T cells.25 Control animals
that were mock-treated or inoculated with HSVlac amplicon virus did not
show any significant CTL responses and appeared tolerized to the tumor.
Because murine T cells respond to costimulation by human CD80 (Fig 3)
and murine T cells migrate in vitro in response to human RANTES (Table
1), we chose to use the human cDNA for the experiments described above.
Furthermore, the CTL response observed to contralateral EL4 cells using
human CD80 and RANTES cDNA suggests that systemic immunity did not
depend on response to xenogeneic or HSV viral antigens. It has been
reported by Townsend et al28 that specificity and longevity
of antitumor responses induced by CD80-transfected tumor cells are
maintained for more than 90 days, and other reports have detected CTL
activity for up to 6 months.13 We measured CTL activity at
different times up to 2 months and significant levels of CTL activity
were sustained in mice that rejected tumors after the intratumoral delivery of HSV amplicon virus (data not shown). After rechallenge with
the parental EL4 tumor cells, we observed an increase in CTL activity
most likely mediated by memory T cells (data not shown). In addition,
the observed CTL activity was mediated predominantly by the
CD8+ CTLs.
Gene therapy strategies have involved numerous approaches. Among the
viral vectors, adenovirus, adeno-associated virus, and retroviruses
have been extensively analyzed. Other investigators have observed
regression of established tumors using adenovirus vectors engineered to
express IL-2.36 Limitations with the use of retroviruses
have been the lower achievable titers and inability to transduce
nondividing cells. HSV vectors have several advantages, including
moderately high titer, robust levels of expression, increased packaging
capability (150 kb), and transduction of multiple copies of the gene of
interest. Dilloo et al37 used replication defective recombinant HSV vectors derived from HSV-2 to transduce the
GM-CSF gene to elicit an antileukemic response against the A20 murine
leukemia cell line in vivo. These investigators also report ability to
withstand challenge with a lower number (105) of leukemic
cells when followed by several subcutaneous injections of an equal
number of cells transduced ex vivo with HSV vectors expressing
GM-CSF.37 Amplicon vectors have several advantages relative
to replication defective HSV as recombination is not necessary for
vector generation. Use of amplicons confers the ability to deliver
multiple genes per infectious event. Amplicon vectors should in theory
be less prone to recombination and regeneration of infectious HSV virus
due to the absence of the viral genome. Further studies with HSV
amplicons will define the potential utility of this vector in humans in
the setting of established HSV-specific immunity and pre-existing
malignancy.
| |
ACKNOWLEDGMENT |
The authors sincerely acknowledge the antibodies for Thy-1, CD3, CD4,
and CD8 received from Dr Edith Lord and Dr Richard Phipps (University
of Rochester Cancer Center, Rochester, NY). Dr Richard Raubertas
(Department of Biostatistics, University of Rochester Medical Center)
is acknowledged for his help in statistical analysis. Karen Rosell from
the Hematology-Oncology Unit was very helpful in the chemotaxis assay.
The author M.K. dedicates this manuscript to the memory of Somnath
Ghosh, MD, PhD, who had been a guiding beacon during his PhD research
work.
| |
FOOTNOTES |
Submitted January 21, 1998;
accepted September 22, 1998.
Supported by National Institutes of Health (NIH) Grant No. AI07285-10
to M.K., NIH Grant No. PO1 CA59326 and the University of Rochester
Cancer Center Discovery Fund to J.D.R., and NIH Grants No. DK 53160 and
NS36420 to H.J.F.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Joseph D. Rosenblatt, MD, University of
Rochester Medical Center, Box 704, 601 Elmwood Ave, Rochester, NY
14642; e-mail: Joe_Rosenblatt{at}urmc.rochester.edu.
| |
REFERENCES |
1.
Green JM, Noel PJ, Sperling AI, Walunas TL, Gray GS, Bluestone JA, Thompson CB:
Absence of B7-dependent responses in CD28-deficient mice.
Immunity
1:501, 1994[Medline]
[Order article via Infotrieve]
2.
Bluestone JA:
New perspectives of CD28-B7-mediated T cell costimulation.
Immunity
2:555, 1995[Medline]
[Order article via Infotrieve]
3.
Mueller DL, Jenkins MK, Schwartz RH:
Clonal expansion versus functional clonal inactivation: A costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy.
Annu Rev Immunol
7:445, 1989[Medline]
[Order article via Infotrieve]
4.
Guinan EC, Gribben JG, Boussiotis VA, Freeman GJ, Nadler LM:
Pivotal role of the B7:CD28 pathway in transplantation tolerance and tumor immunity.
Blood
84:3261, 1994[Abstract/Free Full Text]
5.
Cardoso AA, Schultze JL, Boussiotis VA, Freeman GJ, Seamon MJ, Laszlo S, Billet A, Sallan SE, Gribben JG, Nadler LM:
Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen.
Blood
88:41, 1996[Abstract/Free Full Text]
6.
Schultze JL, Seamon MJ, Michalak S, Gribben JG, Nadler LM:
Autologous tumor infiltrating T cells cytotoxic for follicular lymphoma cells can be expanded in vitro.
Blood
89:3806, 1997[Abstract/Free Full Text]
7.
Gaken JA, Hollingsworth SJ, Hirst WJ, Buggins AG, Galea-Lauri J, Peakman M, Kuiper M, Patel P, Towner P, Patel PM, Collins MK, Mufti GJ, Farzaneh F, Darling DC:
Irradiated NC adenocarcinoma cells transduced with both CD80 and interleukin-2 induce CD4+-mediated rejection of established tumors.
Hum Gene Ther
8:477, 1997[Medline]
[Order article via Infotrieve]
8.
Gribben JG, Cardoso AA, Schultze JL, Nadler LM:
Biologic response modifiers in acute lymphoblastic leukemia.
Leukemia
11:S31, 1997 (suppl 4)
9.
Schall TJ, Bacon K, Toy KJ, Goeddel DV:
Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES.
Nature
347:669, 1990[Medline]
[Order article via Infotrieve]
10.
Bacon KB, Premack BA, Gardner P, Schall TJ:
Activation of dual T cell signaling pathways by the chemokine RANTES.
Science
269:1727, 1995[Abstract/Free Full Text]
11.
Mule JJ, Custer M, Averbook B, Yang JC, Weber JS, Goeddel DV, Rosenberg SA, Schall TJ:
RANTES secretion by gene-modified tumor cells results in loss of tumorigenicity in vivo: Role of immune cell subpopulations.
Hum Gene Ther
7:1545, 1996[Medline]
[Order article via Infotrieve]
12.
Wendtner CM, Nolte A, Mangold E, Buhmann R, Maass G, Chiorini JA, Winnacker EL, Emmerich B, Kotin RM, Hallek M:
Gene transfer of the costimulatory molecules B7-1 and B7-2 into human multiple myeloma cells by recombinant adeno-associated virus enhances the cytolytic T cell response.
Gene Ther
4:726, 1997[Medline]
[Order article via Infotrieve]
13.
Dunussi-Joannopoulos K, Weinstein HJ, Nickerson PW, Strom TB, Burakoff SJ, Croop JM, Arceci RJ:
Irradiated B7-1 transduced primary acute myelogenous leukemia (AML) cells can be used as therapeutic vaccines in murine AML.
Blood
87:2938, 1996[Abstract/Free Full Text]
14.
Hirst WJ, Buggins A, Darling D, Gaken J, Farzaneh F, Mufti GJ:
Enhanced immune costimulatory activity of primary acute myeloid leukaemia blasts after retrovirus-mediated gene transfer of B7.1.
Gene Ther
4:691, 1997[Medline]
[Order article via Infotrieve]
15.
Hodge JW, Abrams S, Schlom J, Kantor JA:
Induction of antitumor immunity by recombinant vaccinia viruses expressing B7-1 or B7-2 costimulatory molecules.
Cancer Res
54:5552, 1994[Abstract/Free Full Text]
16.
Lu B, Federoff HJ:
Herpes simplex virus type 1 amplicon vectors with glucocorticoid-inducible gene expression.
Hum Gene Ther
6:419, 1995[Medline]
[Order article via Infotrieve]
17.
Geller AI, Keyomarsi K, Bryan J, Pardee AB:
An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology.
Proc Natl Acad Sci USA
87:8950, 1990[Abstract/Free Full Text]
18.
Mesri EA, Federoff HJ, Brownlee M:
Expression of vascular endothelial growth factor from a defective herpes simplex virus type 1 amplicon vector induces angiogenesis in mice.
Circ Res
76:161, 1995[Abstract/Free Full Text]
19.
Karpoff HM, D'Angelica M, Blair S, Brownlee MD, Federoff H, Fong Y:
Prevention of hepatic tumor metastases in rats with herpes viral vaccines and gamma-interferon.
J Clin Invest
99:799, 1997[Medline]
[Order article via Infotrieve]
20.
Challita-Eid PM, Penichet ML, Shin SU, Poles TM, Mosammaparast N, Mahmood K, Slamon DJ, Morrison SL, Rosenblatt JD:
A B7.1-antibody fusion protein retains antibody specificity and ability to activate via the T cell costimulatory pathway.
J Immunol
160:3419, 1998[Abstract/Free Full Text]
21.
Johnson PA, Miyanohara A, Levine F, Cahill T, Friedmann T:
Cytotoxicity of a replication-defective mutant of herpes simplex virus type 1.
J Virol
66:2952, 1992[Abstract/Free Full Text]
22.
Paterson T, Everett RD:
A prominent serine-rich region in Vmw175, the major transcriptional regulator protein of herpes simplex virus type 1, is not essential for virus growth in tissue culture.
J Gen Virol
71:1775, 1990[Abstract/Free Full Text]
23.
Tung C, Federoff HJ, Brownlee M, Karpoff H, Weigel T, Brennan MF, Fong Y, Hammes HP, Federoff HJ, Brownlee M:
Rapid production of interleukin-2-secreting tumor cells by herpes simplex virus-mediated gene transfer: Implications for autologous vaccine production: Nerve growth factor prevents both neuroretinal programmed cell death and capillary pathology in experimental diabetes.
Hum Gene Ther
7:2217, 1996[Medline]
[Order article via Infotrieve]
24.
Lundberg A, Gribben S, Lazo S, Nadler LM:
Expression and induction of B7 on leukemic cells isolated from patients with haematologic malignancies.
Blood
82:123a, 1993 (abstr, suppl 1)
25.
Chen L, McGowan P, Ashe S, Johnston J, Li Y, Hellstrom I, Hellstrom KE:
Tumor immunogenicity determines the effect of B7 costimulation on T cell-mediated tumor immunity.
J Exp Med
179:523, 1994[Abstract/Free Full Text]
26.
Li Y, McGowan P, Hellstrom I, Hellstrom KE, Chen L:
Costimulation of tumor-reactive CD4+ and CD8+ T lymphocytes by B7, a natural ligand for CD28, can be used to treat established mouse melanoma.
J Immunol
153:421, 1994[Abstract]
27.
Baskar S, Ostrand-Rosenberg S, Nabavi N, Nadler LM, Freeman GJ, Glimcher LH:
Constitutive expression of B7 restores immunogenicity of tumor cells expressing truncated major histocompatibility complex class II molecules.
Proc Natl Acad Sci USA
90:5687, 1993[Abstract/Free Full Text]
28.
Townsend SE, Su FW, Atherton JM, Allison JP:
Specificity and longevity of antitumor immune responses induced by B7-transfected tumors.
Cancer Res
54:6477, 1994[Abstract/Free Full Text]
29.
Matulonis UA, Dosiou C, Lamont C, Freeman GJ, Mauch P, Nadler LM, Griffin JD:
Role of B7-1 in mediating an immune response to myeloid leukemia cells.
Blood
85:2507, 1995[Abstract/Free Full Text]
30.
Hirano N, Takahashi T, Takahashi T, Azuma M, Yazaki Y, Yagita H, Hirai H:
Protective and therapeutic immunity against leukemia induced by irradiated B7-1 (CD80)-transduced leukemic cells.
Leukemia
11:577, 1997 (suppl 3)
31.
Maric M, Chen L, Sherry B, Liu Y:
A mechanism for selective recruitment of CD8 T cells into B7-1-transfected plasmacytoma: Role of macrophage-inflammatory protein 1alpha.
J Immunol
159:360, 1997[Abstract]
32.
Federoff HJ, Geschwind M, Geller AI, Kessler JA, Brownlee M:
Expression of nerve growth factor in vivo from a defective herpes simplex virus 1 vector prevents effects of axotomy on sympathetic ganglia.
Proc Natl Acad Sci USA
89:1636, 1992[Abstract/Free Full Text]
33.
Dilloo D, Brown M, Roskrow M, Zhong W, Holladay M, Holden W, Brenner M:
CD40 ligand induces an antileukemia immune response in vivo.
Blood
90:1927, 1997[Abstract/Free Full Text]
34.
Dilloo D, Bacon K, Holden W, Zhong W, Burdach S, Zlotnik A, Brenner M:
Combined chemokine and cytokine gene transfer enhances antitumor immunity.
Nat Med
2:1090, 1996[Medline]
[Order article via Infotrieve]
35.
Adema GJ, Hartgers F, Verstraten R, de Vries E, Marland G, Menon S, Foster J, Xu Y, Nooyen P, McClanahan T, Bacon KB, Figdor CG:
A dendritic-cell-derived C-C chemokine that preferentially attracts naive T cells.
Nature
387:713, 1997[Medline]
[Order article via Infotrieve]
36.
Cordier L, Duffour MT, Sabourin JC, Lee MG, Cabannes J, Ragot T, Perricaudet M, Haddada H:
Complete recovery of mice from a pre-established tumor by direct intratumoral delivery of an adenovirus vector harboring the murine IL-2 gene.
Gene Ther
2:16, 1995[Medline]
[Order article via Infotrieve]
37.
Dilloo D, Rill D, Entwistle C, Boursnell M, Zhong W, Holden W, Holladay M, Inglis S, Brenner M:
A novel herpes vector for the high-efficiency transduction of normal and malignant human hematopoietic cells.
Blood
89:119, 1997[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
K. H. Yi, H. Nechushtan, W. J. Bowers, G. R. Walker, Y. Zhang, D. G. Pham, E. R. Podack, H. J. Federoff, K. A. Tolba, and J. D. Rosenblatt
Adoptively Transferred Tumor-Specific T Cells Stimulated Ex vivo Using Herpes Simplex Virus Amplicons Encoding 4-1BBL Persist in the Host and Show Antitumor Activity In vivo
Cancer Res.,
October 15, 2007;
67(20):
10027 - 10037.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Casati, C. Camisaschi, F. Rini, F. Arienti, L. Rivoltini, F. Triebel, G. Parmiani, and C. Castelli
Soluble Human LAG-3 Molecule Amplifies the In vitro Generation of Type 1 Tumor-Specific Immunity.
Cancer Res.,
April 15, 2006;
66(8):
4450 - 4460.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Liu, X. Guan, and X. Ma
Interferon Regulatory Factor 1 Is an Essential and Direct Transcriptional Activator for Interferon {gamma}-induced RANTES/CCl5 Expression in Macrophages
J. Biol. Chem.,
July 1, 2005;
280(26):
24347 - 24355.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Shi, S. Cao, M. Mitsuhashi, Z. Xiang, and X. Ma
Genome-Wide Analysis of Molecular Changes in IL-12-Induced Control of Mammary Carcinoma via IFN-{gamma}-Independent Mechanisms
J. Immunol.,
April 1, 2004;
172(7):
4111 - 4122.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Porosnicu, A. Mian, and G. N. Barber
The Oncolytic Effect of Recombinant Vesicular Stomatitis Virus Is Enhanced by Expression of the Fusion Cytosine Deaminase/Uracil Phosphoribosyltransferase Suicide Gene
Cancer Res.,
December 1, 2003;
63(23):
8366 - 8376.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
P. Ghia, P. Transidico, J. P. Veiga, C. Schaniel, F. Sallusto, K. Matsushima, S. E. Sallan, A. G. Rolink, A. Mantovani, L. M. Nadler, et al.
Chemoattractants MDC and TARC are secreted by malignant B-cell precursors following CD40 ligation and support the migration of leukemia-specific T cells
Blood,
August 1, 2001;
98(3):
533 - 540.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. A. Tolba, W. J. Bowers, S. P. Hilchey, M. W. Halterman, D. F. Howard, R. E. Giuliano, H. J. Federoff, and J. D. Rosenblatt
Development of herpes simplex virus-1 amplicon-based immunotherapy for chronic lymphocytic leukemia
Blood,
July 15, 2001;
98(2):
287 - 295.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Todo, R. L. Martuza, M. J. Dallman, and S. D. Rabkin
In Situ Expression of Soluble B7-1 in the Context of Oncolytic Herpes Simplex Virus Induces Potent Antitumor Immunity
Cancer Res.,
January 1, 2001;
61(1):
153 - 161.
[Abstract]
[Full Text]
|
|
|
|
|
Top
Abstract
Introduction