Affiliations of authors: T. S. Griffith, J. Tian, W. Zhao, T. L. Ratliff, Department of Urology, University of Iowa, Iowa City; M. Kawakita, Department of Urology, Faculty of Medicine, Kyoto University, Japan; J. Ritchey, Washington University, St. Louis, MO; J. Tartaglia, Aventis Pasteur, Toronto, ON, Canada; I. Sehgal, T. C. Thompson, Scott Department of Urology, Baylor College of Medicine, Houston, TX.
Correspondence to: Thomas S. Griffith, Ph.D., Department of Urology, University of Iowa, 200 Hawkins Dr., 428 MRC, Iowa City, IA 522421089 (e-mail: thomas-griffith{at}uiowa.edu).
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ABSTRACT |
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INTRODUCTION |
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Other immunotherapeutic approaches under development rely on viral vectors to transfer genes for tumor-associated antigens or immunostimulatory cytokines into target cells (4,69). Retroviral vectors, however, have been used with limited success (10,11) because these vectors do not infect all cells in a given population, target cells must be proliferating for viral integration, and there is an extended time between infection and expression of the transferred gene. Recombinant vaccinia virus has been used to deliver genes into tumor cells, but its use is limited because it replicates in tumor cells and thus toxic effects are associated with its systemic distribution (12). As an alternative, several poxvirus variants have been identified or developed that possess useful gene transfer qualities. The canarypox virus ALVAC is such a variant that does not replicate in mammalian cells and is an efficient gene-delivery vector (13). When carrying genes for rabies glycoprotein or measles hemagglutinin glycoproteins (1416), ALVAC has proved to be a safe and effective vector in humans and in animals and can induce immune responses and protective immunity against challenge with the cognate pathogens (15,17). These characteristics suggest that ALVAC recombinants may be used for gene delivery in cancer therapy. Previous studies from this laboratory (18) have demonstrated that ALVAC vectors can efficiently insert immunostimulatory cytokine genes into prostate cancer cells and that the genes can produce high levels of the cytokine and thus can induce antitumor activity.
In this study, we used immunostimulatory cytokine genes for granulocytemacrophage colony-stimulating factor (GM-CSF), interleukin 2 (IL-2), IL-12, tumor necrosis factor- (TNF-
), and the highly aggressive and weakly immunogenic RM-1 prostate cancer model. We chose the ALVAC canarypox virus to deliver cytokine genes to RM-1 cells and explored the mechanism of the induced antitumor response.
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MATERIALS AND METHODS |
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The RM-1 mouse prostate tumor model, syngeneic to C57BL/6 mice, was obtained from Dr. Timothy Thompson (Baylor College of Medicine, Houston, TX). RM-1 cells, generated by transduction with the ras and myc oncogenes, produce a poorly differentiated carcinoma when they are implanted into C57BL/6 mice. The cells are cultured in Dulbecco's modified Eagle medium (DMEM) (BioWhittaker, Inc., Walkersville, MD) containing 10% fetal calf serum (FCS) and 10 mM HEPES buffer (pH 7.0). Male C57BL/6 and severe combined immunodeficient (SCID) mice were purchased from Charles River Laboratories (Wilmington, MA) at 68 weeks of age. In all in vivo experiments, groups contained at least five randomly allocated animals, and experiments were repeated at least three times with similar results. All animal experiments were approved by the University of Iowa Animal Care Committees and met all animal care guidelines for the institution.
Antibodies
Monoclonal antibodies (MAbs) against the major histocompatibility complexes (MHCs) Kb, Db, and I-Ab and the costimulatory molecules CD80, CD86, intercellular adhesion molecules ICAM-1 and ICAM-2, Fas, and CD40 and cytokine-reactive antibodies against IL-2, IL-12, GM-CSF, and TNF- for use in enzyme-linked immunosorbent assays (ELISAs) were purchased from PharMingen (San Diego, CA). The anti-CD4 MAb GK1.5 [rat IgG2b (19)], anti-CD8 MAb 2.43 [rat IgG2b (20)], and anti-NK1.1 MAb PK136 [mouse IgG2a (21)] were prepared in the laboratory from ascites fluids generated in SCID mice by precipitating immunoglobulins with 50% ammonium sulfate. After dialysis in phosphate-buffered saline (PBS), MAbs were diluted to 1 mg/mL in PBS.
Flow Cytometry
Flow cytometry was performed as described previously (22). Briefly, cultured tumor cells were harvested with 10 mM EDTA and washed with 10% horse serum in PBS. Cells were incubated with the various unlabeled primary MAbs (10 µg/mL) for 15 minutes on ice. After two washes with 1% FCS in PBS, primary antibody bound to the cells was detected with fluorescein isothiocyanate-conjugated secondary MAb (Sigma Chemical Co., St. Louis, MO; 10 µg/mL for 15 minutes on ice). After two washes with 1% FCS in PBS, unfixed cells were analyzed immediately by flow cytometry on a FACScan (Becton Dickinson, Mountain View, CA). To verify depletion of CD4+ and CD8+ T-cell subsets in vivo, we isolated splenocytes from antibody-treated or untreated C57BL/6 mice and stained them with either the anti-CD4 MAb GK1.5 or the anti-CD8 MAb 2.43, as described above. After two washes with 1% FCS in PBS, primary antibody bound to the cells was detected with fluorescein isothiocyanate-conjugated secondary MAb, as described above. After two washes with 1% FCS in PBS, cells were analyzed by flow cytometry on a FACScan. More than 95% of the CD4+ and CD8+ cells were depleted relative to the numbers present in untreated mice.
Viral Vectors and Infection
ALVAC is a canarypox virus-based vector that is productively replicated only in avian cells (13). Parental ALVAC (ALVAC-par) vector (ALVAC virus Cppp) and ALVAC vectors carrying a murine GM-CSF gene (vCP319), a murine IL-2 gene (vCP275), a murine IL-12 gene (vCP301), and a human TNF- gene (vCP245) were developed at Aventis Pasteur (Toronto, ON, Canada), as described previously (1316,23,24). After infection of RM-1 cells, the levels of proteins produced from the transgene of each vector were evaluated by ELISA.
RM-1 cells were harvested and cultured in DMEM containing 10% FCS and 10 mM HEPES buffer (pH 7.0) the day before infection. For infection with ALVAC, the medium was changed to DMEM with 2% FCS, and ALVACcytokine recombinants were added to the cells in various multiplicities of infection (MOIs). The cells were incubated for 24 hours before the measurement of cytokine production. For tumor therapy studies, cells were incubated with virus for 4 hours, and then the infected cells were removed from tissue culture plates, washed with PBS, resuspended to the desired cell density in PBS, and injected subcutaneously into mice on the flank.
Tumor Inhibition Studies
RM-1 cells infected with one or two of the ALVACcytokine recombinants described above were injected subcutaneously into C57BL/6 or SCID mice. For all experiments, uninfected RM-1 cells or RM-1 cells infected with the ALVAC-par virus served as controls. Tumor outgrowth and animal survival were monitored daily. Results are presented as the percentage of mice with palpable tumors. A palpable tumor had a diameter of at least 23 mm. Mice were killed when tumors reached a diameter of 20 mm or if the animals became moribund or cachectic.
Immunization/Rechallenge Study
RM-1 cells (5 x 105 cells) infected with the indicated ALVAC recombinant(s) were irradiated (70 Gy for 30 minutes) to eliminate the cell's ability to proliferate and thus form tumors; they were then injected subcutaneously into mice on the flank. Ten days later, the mice were challenged with 5 x 105 viable, uninfected RM-1 cells injected subcutaneously on the contralateral flank. Tumor outgrowth was monitored, as described above. Background values were determined by measurment of the response in naive mice challenged with 5 x 105 uninfected RM-1 cells or ALVAC-parental-infected RM-1 cells.
Cytotoxic T Lymphocyte Assays
Splenocytes (composed of red blood cells, T cells, B cells, NK cells, and interstitial cells) were harvested from control or experimental mice, as described above. Red blood cells in the preparation were lysed with ACK lysing buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM Na2EDTA [pH 7.2]), and the remaining cells were cultured with IL-2 (10 U/mL) alone or with IL-2 and mitomycin C (40 µg/mL)-treated RM-1 cells as stimulator cells, at a splenocyte/stimulator ratio of 50 : 1. IL-2 stimulates the proliferation of primed precursor CD8+ cytotoxic T lymphocytes (CTLs) in the splenocyte preparation. After 48 hours, viable splenic effector cells were isolated by centrifugation (800g for 20 minutes at 4 °C) in Ficoll. The effector cells were resuspended in complete medium and incubated with 51Cr-loaded experimental target cells (RM-1 or EL-4 cells) at various cell ratios for 4 hours. All assays were performed in round-bottom 96-well plates. Spontaneous or total release of 51Cr was determined in the presence of medium alone or 1% Nonidet P-40, respectively. The percent specific lysis was calculated as 100 x (experimental counts per minute [cpm] spontaneous cpm)/(total cpm spontaneous cpm). Experimental, spontaneous, and total cpm values used were the mean of triplicate wells, with the standard deviations being less than 10% of the calculated mean for each group, signifying accuracy within each culture condition. The spontaneous release of 51Cr from the target cells never exceeded 20% of maximal 51Cr release.
In Vivo Depletion
The following MAbs were used for the in vivo depletion studies: GK1.5, an anti-CD4 MAb; 2.43, an anti-CD8 MAb; and PK136, an anti-NK1.1 MAb. For the in vivo depletion of various cell populations, MAbs (100 µg/day) were injected into C57BL/6 mice daily for 5 days. Mice were allowed to rest for 2 days and then were given an injection of the same MAbs (100 µg/day) for an additional 5 days. Flow cytometry analysis (CD4+ and CD8+ T cells) and 51Cr-release assays (NK cells) were performed to verify which subset of cells was depleted. To determine antitumor activity in these mice, we shaved a region on the flank of each mouse, injected RM-1 cells subcutaneously at the shaved site, and monitored tumor growth and animal survival over time.
Statistical Analyses
An analysis of differences was performed by Dr. Justine Ritchie (University of Iowa Cancer Center, Iowa City), who used the log-rank test. Survival was estimated by the KaplanMeier method. The 95% confidence intervals (CIs) were based on the normal approximation but constrained to be between 0% and 100%. In experiments where no deaths were observed, an exact 95% one-sided binomial CI was used instead. All other statistical tests were two-sided.
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RESULTS |
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We assessed the ability of RM-1 murine prostate tumor cells to act as immunologic target cells by use of flow cytometry to analyze the expression levels of the following immunologically relevant cell surface proteins: MHC class I and class II antigens, the costimulatory molecules CD80 and CD86, the intercellular adhesion molecules ICAM-1 and ICAM-2, Fas receptor, and CD40. RM-1 cells constitutively expressed MHC class I antigens, and incubation with interferon (IFN
) markedly increased the levels of these antigens (Fig. 1
). In contrast, MHC class II antigens, ICAM-1, ICAM-2, CD80, and CD86 were not detected on RM-1 cells before or after stimulation with IFN
. Fas receptor and CD40 were detected on unstimulated RM-1 cells, and their expression was slightly increased by incubation with IFN
.
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When implanted into immunocompetent mice, RM-1 cells are weakly immunogenic cells that form solid tumors; thus, RM-1 cells are suitable for immunologic gene therapy studies (18). We first determined whether RM-1 cells infected in vitro with the various ALVACcytokine recombinants secreted the corresponding cytokines. The day before infection, RM-1 cells were plated in six-well tissue culture plates at a density of 106 cells per well. The cells were then infected with the ALVACcytokine recombinants ALVACTNF-, ALVACGM-CSF, ALVACIL-2, or ALVACIL-12 at an MOI of 5, and the amount of corresponding cytokine was measured in the culture supernatants 24 hours later by ELISA. These cytokine vectors were chosen because each transgene is known to stimulate antitumor responses. We found that 106 ALVACIL-2-infected cells produced IL-2 at 28.6 ng/mL in 24 hours, 106 ALVACIL-12-infected cells produced IL-12 at 7 ng/mL in 24 hours, 106 ALVACGM-CSF-infected cells produced GM-CSF at 11.2 pg/mL in 24 hours, and 106 ALVACTNF-
-infected cells produced TNF-
at 13 ng/mL in 24 hours. Infection at an MOI greater than 10 resulted in high cell death that was directly related to the high level of viral infection but not to actions of the cytokines, because infection with ALVAC-par at high MOIs was also toxic (data not shown). Levels of transgene-derived cytokines were also measured as a function of time. Five hours after infection, ALVACIL-2-infected RM-1 cells had produced, on average, about 10% the amount of IL-2 (2.9 ng/mL) that they had produced by 24 hours (28.6 ng/mL). Cytokine levels peaked 3 days after infection and were still detected 7 days after infection. Similar results were observed with the other ALVACcytokine vectors (data not shown). Thus, an MOI of 5 produced sufficient levels of cytokine with little associated cell death and was used for all subsequent experiments (18).
For the determination of whether this method of gene transfer affected RM-1 cell growth in vivo, RM-1 cells were infected with the various ALVACcytokine recombinants in vitro. Four hours later, the infected cells were washed and then injected subcutaneously into the flank of male C57BL/6 mice. As shown in Fig. 2, A, mice given an injection of RM-1 tumor cells infected with ALVACIL-12, ALVACGM-CSF, or ALVACTNF-
had a statistically significant survival advantage (P<.001 for each) compared with mice given an injection of RM-1 cells infected with ALVAC-par. In contrast, there was no statistically significant survival advantage among mice given an injection of ALVACIL-2-infected RM-1 cells (P = .102). Mice given an injection of RM-1 cells infected with each ALVACcytokine vector were monitored for 100 days, with no further change in tumor outgrowth. In addition, all mice behaved normally; no mouse demonstrated a behavioral change indicative of cytokine-induced toxicity after the injection of ALVACcytokine-infected RM-1 cells (data not shown).
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Mechanism of Tumor Regression
To determine whether mice receiving RM-1 cells infected with ALVACcytokine recombinants also developed immunity to RM-1 cells on rechallenge, we first immunized mice with -irradiated (70 Gy for 30 minutes) ALVACcytokine (TNF-
/IL-2, TNF-
/IL-12, or TNF-
/GM-CSF)-infected RM-1 cells. The
-irradiated, ALVACcytokine-infected RM-1 cells produced the corresponding cytokines (TNF-
/IL-2, TNF-
/IL-12, or TNF-
/GM-CSF) at levels equivalent to those produced by nonirradiated-infected cells (data not shown). Control mice were immunized with irradiated uninfected RM-1 cells or irradiated ALVAC-par-infected RM-1 cells. Ten days after receiving the irradiated cells, all groups were challenged with normal (i.e., nonirradiated and uninfected) RM-1 cells, and tumor outgrowth was followed. We did not observe increased survival in any group (Fig. 3
). A similar lack of protection against rechallenge was observed in mice that had shown antitumor activity against viable, ALVAC-infected RM-1 tumor cells (data not shown). These results suggest that antigen-specific, T-cell-mediated immunity was not induced against RM-1 tumor cells in these mice.
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Effector Cells Mediating the ALVAC-Induced Anti-RM-1 Response
With the observation that ALVAC-infected RM-1 cells failed to establish tumors without the induction of immunity or activation of antigen-specific CTLs, it was then expected that the absence of the cells responsible for adaptive immunity would not alter the outcome of the cytokine gene therapy procedure. We first explored the immunologic mechanism of the antitumor activity by determining whether T or B cells participated in the antitumor response against the ALVAC-infected RM-1 cells. RM-1 cells infected with various combinations of ALVAC recombinants were injected into SCID (T- and B-cell deficient) mice and C57BL/6 mice, and tumor growth was monitored. We observed that all cytokine combinations inhibited tumor outgrowth equally in C57BL/6 and SCID mice (Fig. 4), suggesting that neither T nor B cells were required for the antitumor effect.
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One possible explanation for the absence of protection against a secondary challenge (shown in Fig. 3) might involve reduced helper T-cell activity, as reported for other systems (6,24). Thus, we attempted to provide the necessary cytokine support for the expression of immunity to a secondary tumor challenge in immunized mice. Mice that had rejected an initial challenge with ALVACTNF-
/ALVACIL-2-infected RM-1 cells or mice immunized with irradiated RM-1 cells were rechallenged with ALVACTNF-
/ALVACIL-2-infected RM-1 cells 50 days after the initial challenge. Naive mice challenged with ALVACTNF-
/ALVACIL-2-infected RM-1 cells or ALVAC-par-infected RM-1 cells served as control subjects. Surprisingly, the growth of ALVACTNF-
/ALVACIL-2-infected RM-1 tumors was not inhibited in mice implanted previously with ALVACTNF-
/ALVACIL-2-infected RM-1 cells (naive mice challenged with ALVAC-par-infected RM-1 cells versus mice given an injection of ALVACTNF-
/IL-2-infected RM-1 cells challenged with ALVACTNF-
/IL-2-infected RM-1 cells; P = .087; Fig. 6
), and rapid tumor outgrowth was observed. In contrast, the expected antitumor effect of ALVACTNF-
/ALVACIL-2 treatment was observed only in the naive mice, suggesting that injection of ALVAC recombinant-infected RM-1 cells actively inhibited the induction of tumor immunity.
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Numerous reports (2635) have described CD4+ T-cell populations that regulate the activation and functions of CD8+ T cells in various settings. Thus, we investigated whether CD4+ T cells contributed to the RM-1 cell-mediated inhibition of CTL activity by injecting mice with ALVACTNF-/ALVACIL-2-infected RM-1 cells and then isolating splenocytes 7 days later. One part of the splenocyte preparation was cultured with RM-1 cells, and the other part was depleted of CD4+ T cells and then cultured with RM-1 cells. Flow cytometry showed that, in the depleted preparations, CD4+ T cells were less than 3% of the population (data not shown). Effector cells from both cultures were harvested after 3 days and tested for lytic activity. Primed, unfractionated splenocytes cultured with IL-2 and RM-1 cells had no lytic activity (see Fig. 7
, C). When CD4+ T cells were depleted before in vitro stimulation, however, cells capable of killing RM-1 target cells but not EL-4 cells (Fig. 7
, D) were observed, suggesting that a CD4+ regulatory T cell was inhibiting RM-1-specific CD8+ CTLs in animals immunized with ALVAC recombinant-infected RM-1 cells.
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DISCUSSION |
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In this article, we demonstrate that genes for several immunostimulatory cytokines transferred into the prostate tumor cells with a canarypox vector before the cells are injected into mice can mediate an antitumor response against a highly aggressive and weakly immunogenic prostate cancer tumor. We found that use of a single type of recombinant ALVAC virus carrying a single cytokine gene (TNF-, IL-2, IL-12, or GM-CSF) was reproducibly, but not statistically significantly, less effective than use of a combination of two types of ALVACcytokine viruses (TNF-
/IL-2, TNF-
/GM-CSF, or TNF-
/IL-12), with the combinations of ALVACTNF-
/ALVACIL-2 or ALVACTNF-
/ALVACIL-12 giving the best results. Inhibition of tumor outgrowth was mediated predominantly by NK cells, with a minor contribution from T cells. Results of in vivo experiments designed to boost the minimal T-cell response suggested that T-cell immunity was being actively suppressed, because a secondary tumor challenge was not inhibited in animals immunized with
-irradiated- or ALVACTNF-
/ALVACIL-12-infected RM-1 cells. Additional experiments identified a CD4+ regulatory cell in splenocyte preparations that inhibited RM-1-specific CTL activity. When splenocyte preparations were depleted of CD4+ cells before co-culturing with RM-1 cells and IL-2, antigen-specific CTL activity reappeared.
ALVAC is a canarypox virus that can infect mammalian cells but cannot replicate in them (13,37), and thus no progeny virus is produced by infected mammalian cells. Indeed, several studies (3840) that examined the consequences of ALVAC infection reported no viral vaccine-associated or virus-induced local or systemic reactions. In contrast, replication-competent recombinant vaccinia virus has also been used as a "live" vaccine, but severe adverse reactions (progressive vaccinia, eczema vaccinatum, and encephalitis) have been reported with its use (12,41). The induction of immune responses and protective immunity against challenge with the cognate pathogen, with essentially no local or systemic reaction against the ALVAC vector, have been reported in studies using ALVAC vectors to carry genes for the rabies virus glycoprotein (14,15), the measles virus fusion and hemagglutinin glycoproteins (16), the feline leukemia virus env and gag proteins (23), and the human immunodeficiency virus-1 envelope glycoprotein (17,24). Thus, ALVAC is a useful tool for gene delivery.
NK1.1+ cells were required for the antitumor response. Depletion of NK1.1+ cells in vivo abrogated the antitumor effects of ALVAC infection. Experiments with SCID mice supported this observation, because the ALVAC combination therapy was equally effective in preventing tumor outgrowth in SCID and in normal mice. Of interest, depletion of either CD4+ or CD8+ T cells consistently reduced the effectiveness of the vaccine in normal mice but not in SCID mice. Enhanced NK cell activity has been reported in SCID mice in other model systems (1,3,5,23), but the reason(s) for the increase in activity are not known. NK cells are the effector cells in the elimination of primary tumor implants [(3,5); this study] and also in CD8-mediated antigen-specific control of metastatic tumor foci and tumor challenge after immunization (3,42). When examined (5,23), tumor cell susceptibility to NK cells was linked to a decrease in MHC class I expression or to an increase in B7 expression. In an investigation of MHC class I modulation of NK cell activity (23), the loss of only a single allele (H-2Kb) was sufficient to induce NK-cell cytolytic activity, and only tumor cells with diminished H-2Kb expression were susceptible to NK cell-mediated lysis. RM-1 cells express moderate levels of H-2Kb, which can be increased by incubation with IFN (Fig. 1
). Because inflammation is induced at the injection site of the ALVAC vaccine (24), perhaps levels of H-2Kb also increase in vivo. Indeed, when ALVACIL-2-infected RM-1 cells were examined after they were injected into mice, the expression of H-2Kb was enhanced (data not shown). Thus, it can be suggested that the ALVACcytokine response is biphasic, where the NK cells respond first to the tumor and then to tumor antigen-specific T cells. The increased NK cell activity in the SCID mice was sufficient to eliminate all of the tumor cells, but "normal" NK cell activity in C57BL/6 mice required a second wave of help from the tumor-antigen-specific T cells to eradicate the tumor.
Animals that received ALVAC-infected RM-1 cells, which lead to the inhibition of tumor outgrowth, had no immunity to a secondary challenge with RM-1 cells. In additional in vitro experiments, tumor-specific CTL activity was not detected when spleen cells from ALVAC vaccine-treated mice were cultured with the antigen (mitomycin C-treated RM-1 cells); CTL activity was observed only when CD4+ T cells were depleted before incubation with the antigen. These results suggest that activation of an immunoregulatory, or suppressor, T-cell population inhibits the activation or proliferation of RM-1-specific CTLs. Although once controversial, the concept of T-cell-mediated suppression of the activation and/or expansion of antigen-specific T cells is now being accepted (26,27). Much of the disbelief in suppressor T cells was largely due to the failure to accurately define a suppressor T-cell lineage, their antigen specificity, and mechanism of action. Recent advances have identified molecular mechanisms by which one population of T cells can regulate another, such as the production of soluble antigen-specific immunosuppressive factors encoded by T-cell receptor and/or
genes (2831). Alternatively, immunosuppression may be induced through cellcell contact in which a noncytolytic negative signal is sent from the regulatory T cell to the target cell. CD4+ CD25+ regulatory T cells have been proposed to function in this manner (3234), by competing for or altering the function or expression of costimulatory molecules on antigen-presenting cells (35). Either of these mechanisms would interfere with the activation or proliferation potential of other T cells responding to antigens expressed on the antigen-presenting cells. The mechanism by which the CD4+ T-cell population regulates the lytic function of CD8+ T cells in our prostate cancer model is under investigation.
ALVAC is a useful means of delivering gene sequences in immunotherapeutic protocols because of its inability to replicate productively in mammalian cells, while retaining high infection efficiency and transgene product expression. The ability of recombinant ALVAC vectors to activate an antitumor response after immunization was demonstrated herein by use of a prostate tumor cell vaccine model. While our data provide further evidence for the feasibility of using recombinant ALVAC vectors in a tumor cell vaccine protocol, studies characterizing the immunoregulatory CD4+ T cells induced in our model system are under way to determine the potential ramifications of activating such a regulatory cell population in clinical patients with the use of this type of tumor cell vaccination protocol.
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NOTES |
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Supported by grant 9884 from the Carver Charitable Trust (Muscatine, IA) and by Public Health Service grant 1R01CA8906201 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.
We thank Dr. David M. Lubaroff (University of Iowa, Iowa City) for his helpful discussions and Linda Buckner and Kris Greiner (University of Iowa) for their expert editorial assistance.
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Manuscript received November 28, 2000; revised April 30, 2001; accepted May 10, 2001.
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