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Immunology |
Department of Life Sciences, Nottingham Trent University, Nottingham NG11 8NS, United Kingdom [S. A. A., S. R., R. C. R.]; Cantab Pharmaceuticals Research Limited, Cambridge CB4 4GN, United Kingdom [C. S. M., M. E. G. B., G. M., C. L. H., E. E., D. M. B., J. G. S.]; Imperial Cancer Research Fund Laboratory of Molecular Therapy, Imperial College of Science and Medicine, Hammersmith Hospital, London W12 0NN, United Kingdom [S. T., R. V.]; Division of Immunology, Queens Medical Centre, Nottingham, United Kingdom [R. A. R.]; and Molecular Medicine Program, Mayo Clinic, Rochester, Minnesota 55905 [R. V.]
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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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, IL-4, tumor necrosis factor 
, GM-CSF, IL-7, or IL-6
enhances antitumor immunity (1, 2, 3, 4, 5, 6)
. In most tumor
models, this results not only in the rejection of the genetically
modified tumor cells but also the induction of systemic immunity
capable of mediating the rejection of a subsequent challenge with
parental, unmodified tumor cells. A major drawback and limitation in using autologous cellular vaccines to treat cancer patients is the need to establish in vitro tumor cell lines prepared from biopsy tumor tissue for the transduction of immuno-modulatory genes. Difficulties associated with establishing cell lines from human tumor biopsy material and the relative inefficiency of many of the transfection methodologies have led to renewed efforts to establish alternative strategies for the efficient delivery of genes into freshly prepared/isolated tumor cells. Vectors that efficiently deliver genes into tumor cells either in vivo or ex vivo are required, and several viral and nonviral vector systems have been investigated for their suitability in this regard (7) . Viral vectors represent the most efficient means of transducing genes into tumor cells, and many replication-competent and replication-defective viruses have been used to deliver genes of interest to in vitro and in vivo targets. HSVs have been used recently for cancer therapy and gene transduction studies. Intratumoral injection of replication-competent attenuated mutants of HSV-1 were shown to be effective in killing malignant gliomas (8 , 9) , and Toda et al. (10) have reported recently that immunization with a defective HSV-1 vector encoding the IL-12 gene in combination with a HSV helper virus can induce local and systemic antitumor immunity to the CT26 murine colon carcinoma. Similarly, systemic therapy using a recombinant adenovirus encoding both subunits of IL-12 inhibited the formation of 3-day hepatic metastasis of murine tumors (11) .
We have reported previously the development of a genetically inactivated HSV-2 vector that is restricted to a single cycle of replication, DISC, for use as a vaccine against genital herpes infection (12, 13, 14) . We have used this virus to deliver cytokine genes to tumor cells, and there are several reasons why this vector is potentially useful for cancer immunotherapy: (a) DISC-HSV-2 is unable to spread from cell to cell; replication of the virus is genetically restricted by deletion of the gH gene, which is essential for the production of infectious progeny; and (b) HSV-2 has a broad host cell range, making the DISC variant an appropriate vehicle for the delivery of genes to a variety of tumors. In addition, DISC-HSV infects nondividing as well as dividing cells and has been shown to rapidly and efficiently infect primary human leukemia and neuroblastoma cells (15) , human carcinoma cells (16) , and cultured murine tumor cells (17) . In the present study, the DISC-HSV-2 vector has been used to deliver genes encoding murine GM-CSF, human IL-2, or the lacZ reporter genes to murine tumor cells and the efficacy of DISC-HSV-2-infected "whole" tumor cell vaccines for prophylactic immunization prior to tumor challenge and for the therapy evaluated in three murine tumor models. The results show that DISC-HSV-2 is an efficient vector for cytokine gene delivery into tumor cells, and that the expression of mGM-CSF or hIL-2 enhances the immunogenicity of whole-cell vaccines. In this study, the therapeutic response was shown to be depend on the functionality of CD4+ and CD8+ lymphocytes.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Female DBA/2, C57Bl/6, BALB/c, and BALB/c
Nu+/Nu+ mice were
purchased from Harlan (UK) Ltd. and were maintained in accordance with
the Home Office Codes of Practice for housing and care of animals.
Infection of Tumor cells with DISC-HSV lacZ Virus.
Tumor cells were either cultured on glass
slides precoated with fibronectin to increase the attachment of the cells or
in 24-well plates at 1 x 105 cells/well and
infected with DISC-HSV lacZ virus at a MOI of 1.25–10 pfu/cell. At
various times postinfection, the cells were either fixed in acetone and
stained for the presence of HSV-2 antigen using a polyclonal anti-HSV-2
antibody (Dako) or fixed in glutaraldehyde and stained for ß-gal by
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside staining
(Promega).
Cytokine Assays.
Expression of cytokines after infection of tumor cells with
DISC-mGM-CSF and DISC-hIL-2 was determined by ELISA (R&D
Systems, United Kingdom). Tumor cells were cultured in 24-well
plates at a concentration of 1 x 105 cells/well overnight. The medium was removed,
and cells were infected with 1.25–10 pfu/cell of each virus in a total
volume of 100–200 µl for 1 h at 37°C. The medium was removed
and replaced by 1 ml of serum-free medium, and the plates were
reincubated at 37°C for various times, up to 48 h. Supernatants
were collected and stored at -20°C and assayed for mGM-CSF or hIL-2.
Apoptosis and HSP Expression.
To determine whether DISC-HSV infection of tumor cells induced cell
death by apoptosis, 5 x 105 cells were
cultured in T25 flasks and infected with DISC lacZ virus at 10 pfu/cell
for 24 h. Floating and adherent (trypsinized) cells were pooled
together for analysis. An ABO-BrdUrd kit from PharMingen (San Diego,
CA) was used according to the manufacturer’s instructions. Briefly,
the cells were prefixed in 1% paraformaldehyde and then stored in 70%
ethanol for 24 h. The cells were then washed and incubated with
terminal deoxynucleotidyl transferase and bromo-dUTP, followed by
FITC-anti- BrdUrd and propidium iodide. DNA breaks are indicative of
apoptosis.
Prophylactic Immunization and Therapy with DISC-HSV-infected
Cells.
Tumor cells were infected with 5–10 pfu/cell with either DISC-mGM-CSF,
DISC-hIL-2, or DISC-lacZ viruses for 1 h. The virus inoculum was
removed, and cells were washed two times in medium. Fresh serum-free
medium was added, and the cells were then irradiated (15,000 rads)
using a Gammacell cesium-137 source; uninfected tumor cells were
prepared in similar manner and used as control. Cells (1 x 106) cells infected with DISC-mGM-CSF or
DISC-hIL-2 viruses (before and after irradiation) were cultured in
24-well plates to assess cytokine production (as detailed above). To
assess the effect of prophylactic vaccination using RENCA cells,
animals were immunized s.c. two times on the right flank at 2-week
intervals with irradiated, noninfected RENCA cells or irradiated,
DISC-infected RENCA cells in a volume of 200 µl (see individual
experiments for details). Unless otherwise stated, animals were
challenged s.c 7 days after the second inoculation, with 5 x 104 (10 times TD50)
parental RENCA cells on the opposite flank.
To assess the efficacy of DISC-HSV infected whole-cell vaccines in therapy, mice received injections in the right flank with 5 x 104 tumor cells and were vaccinated with 1 x 106 DISC-HSV infected-irradiated RENCA cells at the same site or contralaterally on day 0 or 3. Mice then received two additional immunizations at 3-day intervals. Similar protocols using the M3 melanoma and 302R sarcoma were used to confirm the findings obtained with the RENCA model.
Construction of the DISC-HSV Viruses.
Construction of the basic vector DISC-HSV-2 (DISC-HSV) by plasmid
recombination was described previously (17)
. A similar
process was used to construct dH2B (DISC-mGM-CSF), which required a
two-stage recombination strategy. For the first stage, sodium
iodide-purified, wild-type DNA and plasmid DNA (pIMMB56) were
transfected into gH expressing complementing CR1 cells. The plasmid
pIMMB56 contains the lacZ gene under the control of the SV40
promoter; the expression cassette is flanked by HSV sequences to enable
recombination into viral genome and is similar in construction to
pIMMB47+ (17)
. The resulting virus is designated dH2D. For
the second stage, sodium iodide-purified dH2B viral DNA and plasmid DNA
pIMR3 were transfected into CR1 cells as described above. Plasmid pIMR3
was constructed by ligation of the mGM-CSF gene from plasmid
pJL3.2 (received as a gift; Ref. 18
) into the
shuttle vector pIMMB46. Plasmid pIMMB46 had been adapted previously to
contain the CMV promoter and bovine growth hormone poly(A) addition
signal from the plasmid PPRC/CMV (R&D Systems). The resulting virus was
passaged three times on BHK gH+/TK- cells in the presence of
methotrexate to select for TK+ virus. The final virus was designate
dH2B.
dH2J (DISC hIL-2) was constructed by ligation. This process is simpler than traditional recombinant techniques and yields a high frequency of recombinant viruses. A linker sequence containing a unique PacI restriction site was engineered into the basic vector during construction to allow subsequent manipulation. dH2J was constructed by a ligation process similar to basic plasmid construction. The IL-2 gene was excised from the plasmid BBG30 (R&D systems); the plasmid was engineered to construct the native signal sequence and insert a Kosak consensus sequence upstream of the gene. The modified IL-2 gene was ligated into the "Pac ligation" vector (pIMJ2), which contains two PacI sites, before insertion into the virus. The vector pIMJ2 was digested with PacI to release a fragment containing the modified IL-2 gene downstream of the CMV promoter. The expression cassette was ligated with PacI-digested HSV in a similar process to basic plasmid construction. The ligated DNA was transfected into CR2 cells, and the resulting virus was designated DH2J.
Depletion of CD4+ and CD8+ Cells.
The effect of the in vivo depletion of
CD4+ or CD8+ T-cells or
both CD4+ and CD8+ T-cells
on the therapeutic efficacy of the DISC vaccine was investigated.
Groups of 10 mice were given three i.p. injections of 1 mg of anti-CD4
(YTS191.1.2), anti-CD8 (YTS 169.4.2.1), control isotype antibody
(YTH24), or a combination of anti-CD4+ and
anti-CD8+antibodies over a period of 1 week
(19)
. Ten days after the last injection, three
representative animals from each group were tail bled to determine the
efficiency of depletion by flow cytometric analysis of the blood cells
using anti-CD4 and anti-CD8 antibodies (Serotec Ltd. Oxford, United
Kingdom). Mice received injections of RENCA cells (5 x 104 cells/mouse) 7 days after the last antibody
injection. Three vaccinations, with irradiated DISC-infected RENCA
cells, were given 3 days apart at an adjacent body site, commencing on
day 3 after the injection of tumor cells.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The
infectability of RENCA cells using a MOI ranging from 0.3 to 10
pfu/cell was determined. A MOI of 1.25 pfu/cell resulted in 50% of the
cells staining for ß-gal 24 h after infection, whereas a MOI of
2.5 pfu/cell or greater resulted in ß-gal protein expression in
virtually all of the cells (Fig. 1B)
3 h after
infection (Fig. 1C)
. Similar results were obtained for 302R
and M3 cells (results not shown).
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, which was the maximum amount detected in this time
course. The mGM-CSF release from RENCA cells increased proportionally
with virus MOI (0.3 to 10 pfu/cell). Similar results were obtained for
RENCA cells infected with DISC-hIL-2 virus, with the maximum release
occurring 48 h after infection (Fig. 2B)
. For RENCA
cells infected with DISC-hIL-2 virus at a MOI of 10 pfu/cell, 548 x
pg/ml/105 cells of hIL-2 was released into the
supernatant at 24 h. In comparison, the mouse sarcoma 302R cells
released 440 pg/ml/105 cells 24 h after
infection with 10pfu/cell of DISC-hIL2, and the melanoma cell line M3
released 600 pg/ml/105 cells at 24 h.
Cytokine production and release into culture medium by
DISC-HSV-infected cells peaked at 24 and 48 h for mGM-CSF and
IL-2, respectively, and thereafter and by day 5 declined to negligible
levels; this was demonstrated using transwells when culture medium was
replaced with fresh medium at 24-h intervals (results not shown). The
level of cytokine production by DISC-HSV-2-infected tumor cells is high
in comparison to conventional gene transfection is transitional but of
limited duration (72 h) because of the cytopathic effect of the virus.
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, whereas 302R cells showed an increase in the amount of mGM-CSF
release of 
65% and M3 cells a decrease of 40% (results not
shown). A significant (P 
0.01) increase in
hIL-2 release occurred after irradiation of DISC-IL-2-infected RENCA
cells (Table 1
; mean of three experiments). Treatment of RENCA cells
with the protein synthesis inhibitor cycloheximide resulted in a >95%
inhibition of the reporter (LacZ) and cytokine gene expression (results
not shown), confirming that virus replication is essential for gene
expression.
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. Analysis of the DNA profile of cells showed that infected cells
contained more DNA than uninfected cells, indicative of a block in the
S or G2-M phases of the cell cycle, together with
an increase in sub-G1 cells, which is suggestive
of an increase in necrotic cell death (Fig. 3)
. Furthermore, infecting
RENCA cells with DISC-lacZ did not induce the expression of the HSP
protein HSP70, as shown by quantitative mRNA expression (results not
shown), inferring that virus infection does not induce the early onset
of stress response proteins that has been reported previously to
influence the immunogenicity of tumor cells (20)
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. The degree of protective immunity was increased after immunization
with 1 x 104 DISC-infected cells
expressing mGM-CSF compared with nonimmunized control mice or mice
receiving irradiated RENCA cells or irradiated RENCA cells infected
with DISC-lacZ (Fig. 4A)
. In addition, mice immunized with
1 x 105 DISC-mGM-CSF-infected
RENCA cells showed an increased level of protection to tumor challenge
compared with mice immunized with irradiated (noninfected) RENCA cells
and control (nonimmunized) mice (Fig. 4B)
. It was observed
that immunization with 1 x 105
irradiated RENCA cells infected with DISC-lacZ could also increase
resistance to tumor challenge compared with nonimmunized control mice,
although the degree of protective immunity was consistently lower than
that observed for mice immunized with DISC-mGM-CSF-infected cells.
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, respectively). Vaccination with DISC-hIL-2-infected RENCA
cells gave results that were similar to those obtained using
DISC-mGM-CSF-infected RENCA cells (results not shown). These
experiments were repeated in a second tumor model. Prophylactic
immunization with irradiated DISC- mGM-CSF- or DISC-hIL-2-infected 302R
cells enhanced immunity to tumor challenge and confirmed the results
obtained using the RENCA model (results not shown).
Tumor Therapy Using DISC-HSV-infected Tumor Cells.
The ability of DISC-infected tumor cells to influence the growth
of established tumors was investigated in three tumor models: RENCA,
302R, and M3. Groups of 10 mice were injected s.c. on the right flank
with 5 x 104 (10 x TD50) viable RENCA cells prior to
vaccination with irradiated DISC-infected or irradiated noninfected
RENCA cells. Three immunizations (each containing 1 x 106 irradiated tumor cells) were given s.c. on
the same or contralateral flank on days 3, 6, and 9, and the tumor
incidence and tumor size were recorded during a 9-week observation
period. By day 3 after inoculation of live tumor cells, defined tumor
foci were detected by H&E histological analysis (results not shown),
and for most of the experiment performed, this was the start date for
initiation of therapy. The results (Fig. 5A)
demonstrate a slight but insignificant delay in the onset
of tumors in mice receiving the irradiated (noninfected) RENCA cell
vaccine compared with control mice; this difference was not apparent
when the average tumor sizes of the groups were compared (results not
shown). Mice receiving DISC-mGM-CSF-infected RENCA cells showed a
significant delay in the onset of tumors, and a high proportion of mice
remained tumor free up to 9 weeks after challenge. In addition,
vaccination with DISC-hIL2-infected cells significantly inhibited tumor
growth; 60% of mice remained free of tumor throughout the observation
period (Fig. 5A)
. These results were reproducible and
occurred when immunization was initiated on day 0 or day 3 after tumor
cell inoculation. Up to 80% of mice immunized with either DISC-mGM-CSF
or DISC-hIL-2 vaccines remained free of tumor throughout the study
period (data from several experiments, not shown).
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model. Vaccination, beginning on day 0 after live tumor
cell implantation, with DISC-mGM-CSF or DISC–hIL-2-infected M3 cells
inhibited tumor growth in 60 and 20% of vaccinated animals,
respectively (Fig. 5C)
, and caused a delay in tumor growth
in the remaining mice (results not shown). Inhibition of established M3
tumor growth also occurred in 40% of mice treated with DISC-lacZ
M3-infected cells.
One important feature of this immunotherapy model was the development
of local immunity in vaccinated mice. Vaccination at a site adjacent to
the tumor implantation site with the DISC-mGM-CSF RENCA cell vaccine
was effective in delaying the onset and growth of tumors; however,
immunization on the contralateral flank was less effective (Fig. 5C)
. These results demonstrate that an increased therapeutic
benefit can be derived by local administration of tumor cells infected
with DISC-HSV-2 engineered to express either mGM-CSF or hIL-2 and were
confirmed using the 203R tumor model (results not given).
Vaccine Therapy in T-Cell-deficient Mice.
To determine that T-lymphocytes were required for effective
immunotherapy using DISC whole tumor-cell vaccines, BALB/c nude mice
(Nu+/Nu+) received
injections s.c. with 10 x TD50 of
RENCA cells on the right flank and vaccinated (starting on day 0) three
times (3 days apart) on the same flank with irradiated noninfected
RENCA cells or irradiated DISC-mGM-CSF-infected RENCA cells. The tumor
incidence and growth rate were similar in control and vaccinated
Nu+/Nu+ mice,
indicating that T lymphocytes play a pivotal role in promoting tumor
rejection (results not given). To establish the involvement of
CD4+ and CD8+ T lymphocytes
in immunotherapy, mice were depleted of the respective T-cell
populations by the administration of Mabs raised to either CD4 or CD8
antigens. Seven days after antibody treatment, mice were injected with
5 x 104 RENCA cells and vaccine
therapy (irradiated RENCA cells infected with DISC-mGMCSF) given on
days 3, 6, and 9. The results demonstrate that CD4, CD8, and CD4/CD8
"knock out" mice failed to respond to whole-cell vaccine therapy,
whereas mice inoculated with isotype control serum or untreated mice
were successfully treated by vaccination with DISC-mGM-CSF-infected
RENCA cells (Fig. 6)
. The abrogation of therapeutic efficacy was proportional to the
reduction in the subpopulations of T lymphocytes; the administration of
CD8 Mab caused a 50% depletion of circulating CD8+ T cells and
abrogated the effect of immunotherapy in 60% of mice. Administration
of CD4 Mab reduced circulating CD4+ T cells by <95% and completely
abrogated the effect of vaccine therapy. Collectively, these results
demonstrate an absolute requirement for both CD4+
and CD8+ T lymphocytes for effective
immunotherapy using DISC-mGM-CSF-infected whole-cell vaccination.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We previously constructed gH-deleted HSV-2 to be used as a vaccine for the prevention of HSV-induced disease. This virus, which we term DISC, can only complete one replication cycle in normal cells and was shown to stimulate broad humoral and cell-mediated antiviral immune responses (12) . DISC-HSV offers advantages as a vector system for gene transfer; they are safe because they are unable to spread from cell to cell within the patient, and they have a broad host cell range, making them suitable for the delivery of genes to a variety of tumors. Initial studies have shown that the DISC-HSV-2 will infect a wide range of murine and human tumor cells, including primary human leukemia and neuroblastoma cells (15 , 16 , 22) . Here we show that DISC-HSV is able to infect murine carcinoma, sarcoma, and melanoma cells.
We have used three murine tumors, RENCA, 302R, and M3, as models to assess the ability of DISC-HSV-2 to deliver cytokine genes into tumor cells and undertaken preclinical evaluation of whole-cell vaccines expressing the cytokines mGM-CSF or hIL-2 to assess their ability to promote protective and therapeutic immunity. The RENCA tumor model was used extensively in this study, and the results were confirmed using 302R and M3 tumors. The relationship between the expression of the reporter ß-gal gene and viral proteins after in vitro infection with the DISC-HSV-lacZ virus was established by dual staining for the expression HSV glycoprotein and the ß-gal protein. Infection and reporter gene expression were time and dose dependent. A correlation between ß-gal expression and the MOI was shown and confirmed that the virus was incapable of lateral spread. In a study by Lowstein et al. (23) , recombinant HSV type 1 mutant tsk vectors containing ß-gal were shown to infect neurocortical cells; ß-gal expression was directly related with the MOI of the virus.
Infecting RENCA cells with DISC-HSV-2 encoding mGM-CSF and recombinant hIL-2 genes resulted in the release of cytokines into the culture supernatant in a time-dependent manner. After infection with DISC-HSV-ß-gal, an increase in necrotic cell death versus apoptosis occurred. RENCA cells undergoing death by necrosis may provide addition activation of the immune system in vivo by promoting tumor antigen processing and presentation by professional antigen-presenting cells, leading to an increase in T-cell activation (24 , 25) . In situ killing of tumor cells using suicide gene transfer to induce cell death through a nonapoptotic pathway is associated with enhanced immunogenicity and may in some cases require the induction of HSP expression (20) , although in the present study RENCA cells infected with DISC-HSV failed to show elevated expression of HSP.
On the basis of these in vitro results and because of the potential of these cytokines to activate effector T cells (26) , DISC-mGM-CSF and DISC-hIL-2 vectors were chosen for in vivo studies. In animal models, mGM-CSF expression by tumor cells results in potent systemic antitumor immunity, which can potentiate the rejection of weakly immunogenic murine tumors (27) . IL-2 is also a potent mediator of antitumor immunity and can promote CTL activation and T-cell differentiation, enhance the activation status of natural killer cells, and induce lymphokine-activated killer cell activity (28 , 29) . Interestingly, hIL-2 production and release by DISC-hIL-2-infected cells were significantly increased after irradiation, an effect observed previously by Simova et al. (30) , where administration of irradiated IL-2-secreting plasmacytoma cells was shown to be more effective than nonirradiated cells in promoting tumor immunity. Here, we demonstrate that immunization with irradiated RENCA cells infected with DISC encoding either mGM-CSF or hIL-2 cytokine genes protects mice against challenge with parental tumor cells in a dose-related manner.
DISC-mGM-CSF vaccine therapy prevented tumor growth in a high percentage of mice. The response to therapy was T-lymphocyte dependent and required the participation of both CD4+ and CD8+ T lymphocytes. One important consideration in this therapy model is the relative contribution of HSV infection versus cytokine production. Partial protection was observed after immunization with tumor cells infected with the DISC ß-gal virus (used as a control for cells expressing cytokine), indicating that protection against tumor challenge may, in part, be a consequence of viral infection of the tumor cells; immunization with the ß-gal protein alone does not illicit a measurable antitumor immune response (10) . We suggest that DISC-HSV infection can act as an additional stimulus to enhance the immunogenicity of the tumor cells. HSV infection of mice has been shown to lead to the up-regulation of IL-12 expression (31) and to have potent antitumor effects in animal models (32) , most probably by promoting a Th1 response to tumor antigen(s). Preexisting immunity to HSV infection did not seem to affect the efficacy of this vaccine because no significant difference was shown when animals were preimmunized with HSV prior to tumor implantation and subsequent therapy (data not shown). Irradiated RENCA cell vaccination also induced a degree of protection against rechallenge with the parental tumor line. These data are consistent with previous observations demonstrating that RENCA cells are weakly to moderately immunogenic (33) , and where irradiation itself may affect the immunogenicity of tumor cells through the up-regulation of H-2Kd class I MHC antigens (34) . For the reasons outlined, there is a precedent for using DISC-HSV to deliver immune response genes to tumor cells, in the present study by ex vivo infection of the tumor cells, but additionally by direct in vivo injection into the tumor using a murine colon carcinoma model, where 40% of tumors regressed completely (16) ; we have shown that both approaches are efficacious for therapy in animal models.
GM-CSF has been used to potentiate antitumor immunity by promoting the maturation and function of professional antigen-presenting cells (35 ) and by recruiting additional antigen-specific and nonspecific effector cells. One noticeable feature of the immune response after inoculation of whole-cell vaccines expressing GM-CSF is the prevalence of a delayed-type hypersensitivity response at the site of vaccination and at the site of tumor rejection. Infiltration of tumors by eosinophils in response to GM-CSF has been reported in preclinical and clinical studies (27 , 36) , and a similar response is also observed after immunization with IL-4 gene-transduced vaccines (37 , 38) . There is also evidence that patients treated with an autologous GM-CSF gene-transduced vaccine can undergo an objective clinical response (36) , although it is unclear to what extent eosinophils and effector cells other than CD8+ and CD4+ lymphocytes actually contribute to tumor rejection. Eosinophil infiltration of small established RENCA tumors occurs within 24 h after vaccine therapy with irradiated DISC-mGM-CSF RENCA cells4 and may represent a response associated with the production of Th2 cytokines IL-4 and IL-5 (36) . In conclusion, the results obtained in this study allow us to propose a clinical approach to cancer immunotherapy based on the use of a novel DISC-HSV vector for the efficient delivery of cytokine genes to tumor cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Funded by Cantab Pharmaceuticals. 
2 To whom requests for reprints should be
addressed, at Department of Life Sciences, Nottingham Trent University,
Clifton Lane, Nottingham NG11 8NS, United Kingdom.
3 The abbreviations used are: IL, interleukin;
hIL, human IL; CSF, colony-stimulating factor; GM-CSF,
granulocyte-macrophage CSF; mGM-CSF, murine GM-CSF; HSV, herpes simplex
virus; DISC, disabled infectious single cycle; pfu, plaque-forming
unit(s); HSP, heat shock protein; BrdUrd, bromodeoxyuridine;
CMV, cytomegalovirus; ß-gal, ß-galactosidase; MOI, multiplicity of
infection; TD50, tumor dose 50%; Mab, monoclonal
antibody; gH, glycoprotein H.
Received 8/23/99. Accepted 12/16/99.
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