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Clinical Cancer Research Vol. 11, 735-742, January 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Sequential Immunogene Therapy with Interleukin-12– and Interleukin-15–Engineered Neuroblastoma Cells Cures Metastatic Disease in Syngeneic Mice

Michela Croce1, Raffaella Meazza2, Anna Maria Orengo1, Luana Radic'1, Barbara De Giovanni3, Claudio Gambini3, Barbara Carlini4, Vito Pistoia4, Lorenzo Mortara5, Roberto S. Accolla5, Maria Valeria Corrias4 and Silvano Ferrini1

1 Laboratory of Immunopharmacology, Istituto Nazionale per la Ricerca sul Cancro; Laboratories of 2 Clinical and Experimental Immunology, 3 Pathology, and 4 Oncology, Gaslini Institute, Genoa, Italy and 5 Department of Clinical and Biological Sciences, School of Medicine, University of Insubria, Varese, Italy

Requests for reprints: Silvano Ferrini, Laboratory of Immunopharmacology, Istituto Nazionale per la Ricerca sul Cancro, Largo Benzi 10, 16132 Genoa, Italy. Phone: 39-010-5737372; Fax: 39-010-5737374; E-mail: silvano.ferrini{at}istge.it.


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: To investigate the potential synergistic effects of Neuro2a neuroblastoma cells engineered with IL-12 and/or IL-15 genes in improving survival of syngeneic mice bearing neuroblastoma metastatic disease.

Experimental Design: Neuro2a cells engineered with interleukin (IL)-12 (Neuro2a/IL-12), IL-15 (Neuro2a/IL-15), or both cytokines (Neuro2a/IL-12/IL-15) were injected s.c. in syngeneic A/J mice challenged i.v. with Neuro2a parental cells (Neuro2apc) using different schedules of administration in either preventive or therapeutic settings.

Results: A single injection of Neuro2a/IL-12 or Neuro2a/IL-15 cells induced resistance to a subsequent i.v. Neuro2apc challenge in 45% and 28% of mice, respectively. Neuro2a/IL-12/IL-15 cells protected 28% of mice, showing no synergistic effect. However, sequential vaccination with Neuro2a/IL-12 (day –30) followed by Neuro2a/IL-15 (day –15) protected 71% of mice from subsequent challenge with Neuro2apc. A single dose of Neuro2a/IL-12 prolonged the mean survival time of mice bearing established metastatic neuroblastoma from 21 ± 3 to 46 ± 27 days but failed to cure mice, whereas Neuro2a/IL-15 or Neuro2a/IL-12/IL-15 were ineffective. However, sequential vaccination with Neuro2a/IL-12 (day +3) followed by Neuro2a/IL-15 (day +13) cured 43% of mice as assessed by histologic analysis of different organs from long-term surviving mice. CTL activity against Neuro2apc cells was observed in splenocytes from treated mice, and CD8+ T-cell depletion abrogated the therapeutic effect of vaccination.

Conclusions: Sequential vaccination with IL-12- and IL-15-engineered neuroblastoma cells induced optimal preventive and therapeutic effects, which may be related to the Th1 priming effect of IL-12 followed by the enhancement of CD8+ T-cell responses and their maintenance mediated by IL-15.

Key Words: Cytotoxic T-lymphocytes • neuroblastoma • CD8+ T cells • transfection • cytokines


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuroblastoma is an extracranial tumor of childhood deriving from the neural crest. Neuroblastoma has a broad spectrum of clinical presentation varying from aggressive disease (stage 4) to spontaneous maturation and even regression. Prognosis depends on age and stage as well as on genetic features such as MYC-N amplification (1, 2). Conventional therapy for stage 4 neuroblastoma patients, based on chemotherapy, surgery, and autologous hematopoietic stem cell transplant, allows survival rates of ~20% at 5 years (3); therefore, the search for new therapeutic approaches is warranted.

Gene transfer of immunostimulatory cytokines in tumor cells has been considered an attractive tool to induce immune responses against the tumor cause of the paracrine adjuvant effects of tumor-released cytokines in the absence of systemic toxic effects (4, 5).

Interleukin (IL)-12 is a heterodimeric cytokine with pleiotropic functions, acting as a potent inducer of Th1 responses and as a stimulator of natural killer (NK) cell proliferation and cytotoxicity (6). IL-12 has shown a potent antitumor activity either as a recombinant cytokine (7, 8) or as fusion protein (9) or in gene transfer approaches (10, 11). The antitumor effects of IL-12 have been related at least in part to the IFN-{gamma}-dependent induction of the antiangiogenic CXC chemokines IP-10, MIG, and I-TAC (12–14), which interact with a CXCR-3 isoform expressed on endothelial cells (15). IL-12-engineered tumor cells are highly effective as prophylactic vaccines, because they can protect mice from tumor rechallenge (10) or prevent spontaneous tumorigenesis in mice transgenic for the HER-2/ neu oncogene(16). Nonetheless, in most experimental tumor models, recombinant IL-12- or IL-12-modified tumor cells displayed only a limited activity in the immunotherapy of established tumors (10, 16). Synergistic antitumor effects were reported by the combined use of IL-12 and IL-15 either as recombinant cytokines or in gene transfer approaches in different syngeneic tumor models (17–19). In addition, synergistic effects of cotransduced IL-12 and IL-15 were also reported in syngeneic IFN-{gamma}–/– mice (20) and in nude mice bearing xenografts of human MHC class I–negative lung cancer cells (21). IL-15 is a four {alpha}-helix-bundle cytokine, which stimulates the functional activities of T, B, and NK cells (22, 23). These activities are mediated through a specific IL-15 receptor (R){alpha} chain required for high affinity binding and by the promiscuous IL-2 receptor ß/{gamma} complex involved in signaling (24). The study of IL-15 or IL-15 receptor {alpha} knockout mice revealed an essential role of IL-15 in NK and NK-T-cell development and in the maintenance and function of CD8+ memory T cells (25, 26) . Thus, IL-15 has been used in immunogene therapy of experimental tumors with promising results (19, 27). Responses triggered by IL-15-modified tumor cells involved T and/or NK cells and required IFN-{gamma} as secondary mediator (19, 20, 27).

In this report, we have investigated the potential synergistic effects of murine neuroblastoma cells expressing IL-12 and/or IL-15 in the immunoprevention or immunotherapy of metastatic syngeneic neuroblastoma. Although coexpression of the two cytokines failed to show synergistic effects, the use of a sequential protocol with IL-12-modified cells for priming, followed by IL-15-modified cells for boosting and maintenance of the response, produced enhanced protection in the preventive setting. More importantly, a strong therapeutic effect was observed by this sequential protocol, leading to a 43% cure rate of established neuroblastoma metastases. These effects strictly required CD8+ cells displaying CTL activity against parental neuroblastoma cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Line, Transfection Procedure, and Analysis of Transfectants. Neuro2a (CCL131, American Type Culture Collection, Rockville, MD) and YAC cell lines (kindly provided by Dr. M.P. Colombo, Milan, Italy) were grown in DMEM or RPMI 1640 supplemented with 10% FCS, 2 mmol/L glutamine, penicillin, and streptomycin (BioWhittaker, Walkersville, MD) in a 5% CO 2 atmosphere at 37°C.

Neuro2a parental cells (Neuro2apc) were transfected with 6 µg pmuIL-12IRES1-hygro and/or pVkL/IL-15IRES1-neo (21) or with the empty vectors by the use of FuGene reagent (Roche Biochemicals, Milan, Italy). Stable transfectants were selected in culture medium containing G418 (500 µg/mL) and/or hygromycin (250 µg/mL, Roche) and cloned by limiting dilution. A commercial ELISA kit (Bender MedSystems Diagnostics GmbH, Vienna, Austria) and a proliferation bioassay (27) were used to determine mIL-12 and IL-15 production, respectively (Table 1).


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  		Table 1 IL-15 and IL-12 production by the engineered Neuro2a selected clones

 
Animal Model, Experimental Conditions, and Depletion Procedure. Five-week-old female A/J mice were purchased from Harlan Italy (San Pietro al Natisone, Italy). The animals were housed in pathogen-free colony, and experiments were performed according to the National Regulation on Animal Research Resources and were approved by the Review Board of the Istituto Nazionale per la Ricerca sul Cancro. Groups of 7 to 14 mice were injected using a 25-gauge needle with 1 x 10 6 cells in a volume of 0.1 mL medium either i.v. in the tail vein or s.c. in the flank. All the experiments were repeated twice with identical results. Cell viability was checked before injection in mice and always found >95%.

Depletion studies were done by i.p. injection of rabbit anti-asialo-GM1 antiserum (Wako Chemicals GmbH, Düsseldorf, Germany; 0.1 mL of a 1:10 diluted stock solution/dose) or anti-CD8 (2.43) or anti-CD4 (GK1.5; both from American Type Culture Collection, Rockville, MD) rat monoclonal antibodies (mAb; 100 µg per dose) as reported previously (27). Depleting antibodies were given at day +1, +4, +8, and +14 from Neuro2apc i.v. challenge. Statistical analysis was done by the log-rank or Mann-Whitney test.

Histopathologic Analysis and Reverse Transcription-PCR Analysis for Tyrosine Hydroxylase. All the different organs, as well as the spine and the anterior and posterior leg bones, were systematically removed from killed mice and fixed for 48 hours in formalin. Bones were then decalcified with Decal (DAKO SPA, Milan, Italy) for 2.5 hours. All the tissues were processed for paraffin embedding, sectioned at 6 µm, and stained with H&E.

Total RNA was extracted from bone marrow cells flushed from the two posterior leg bones of the long surviving mice (killed at day 120 post–i.v. injection) using the RNeasy kit (Qiagen, Cologne, Germany) according to the procedure recommended by the manufacturer. Total RNA (1 µg) was then reverse transcribed by employing the first-strand cDNA synthesis kit (Clontech, Palo Alto, CA). cDNA (10 µL) was separately amplified, in a final volume of 50 µL, with 2.5 IU Taq gold polymerase (Applied Biosystems, Foster City, CA) using primers specific for the housekeeping gene GAPDH (supplied in the cDNA synthesis kit) or the murine TH gene. First tyrosine hydroxylase PCR reaction (2 µL) was then amplified with primers internal to those used in the first round. The amplification products were then analyzed on 2% agarose gel stained with ethidium bromide. Primer sequences for first round and nested PCR for murine tyrosine hydroxylase as well as amplification profiles were as described (28).

Fluorescence-Activated Cell Sorting Analysis. Cells were analyzed for MHC class I expression by indirect immunofluorescence and cytofluorimetric analysis using the 34.1.25 (anti-H2K d/D d) mAb. FITC-conjugated goat anti-mouse antiserum (Jackson Labs, West Grove, PA) was used as a second-step reagent. Samples were analyzed by a FACScan analyzer (Becton Dickinson, Milan, Italy).

Mixed Lymphocyte Tumor Cells, CTL Activity Assay, and Cytokine Production Assay. Spleen cells from mice that had rejected Neuro2a cell challenge were restimulated in vitro for 5 days with irradiated Neuro2a cells (20,000 rad) at 50:1 responder/stimulator cell ratio. Cultures were supplemented with IL-2 (25 IU/mL). The capability of lymphoblasts to lyse parental target cells was evaluated by a standard 51Cr release assay, and percentage of lysis was then calculated. Inhibition of lysis was done by adding 1 µg/mL anti-CD3 mAb (14.52.C11) or an irrelevant isotype-matched immunoglobulin to the assay.

Supernatants of splenocytes co-cultured with irradiated Neuro2a cells for 3 days without the addition of recombinant IL-2 were analyzed for IFN-{gamma} production by ELISA using a commercial available kit (Bender MedSystems Diagnostics).

Enzyme-linked immunospot was done on splenocytes from either naive or vaccinated mice. Multiscreen-IP plates (Millipore, Bedford, MA) were coated overnight with 10 µg/mL of either anti-IFN-{gamma} or anti-IL-4 in PBS (both Endogen, Woburn, MA). Plates were then washed with PBS and blocked for 4 hours with PBS-2% bovine serum albumin. Splenocytes were then seeded at 2 x 10 5 cells per well in duplicate in the presence or absence of irradiated Neuro2apc (2 x 10 4 cells per well). After 36 hours, plates were washed with PBS-0.05% Tween 20 and incubated with biotinylated second mAb to IFN-{gamma} or IL-4 (1 µg/mL, Endogen) in PBS-1% bovine serum albumin for 3 hours. Then, horseradish peroxidase–conjugated streptavidin (1:5,000) was added for other 2 hours. After washing, the plates were stained with AEC staining kit (Sigma, St. Louis, MO) and spots were counted using a stereomicroscope. Cell depletion was done by incubation with anti-CD4 or anti-CD8 mAb for 1 hour at 4°C followed by incubation with rabbit complement (Cedarlane, Hornby, Ontario, Canada) for 1 hour at 37°C.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation and Characterization of Genetically Modified Neuro2a Cells. I.v. injection of 10 6 Neuro2a parental cells (Neuro2apc) in syngeneic A/J mice produced a metastatic disease leading to 100% death within 27 days (mean 21 ± 3 days), whereas s.c. injection produced localized tumors in 50% of mice (data not shown). On i.v. injection, Neuro2a cells spread to different organs, closely resembling human stage 4 disease (29, 30), and bone marrow infiltration was detected in all mice by reverse transcription-PCR analysis of tyrosine hydroxylase mRNA expression. Thus, the i.v. injection of Neuro2apc was selected as disease model to test the efficacy of vaccination approaches.

Neuro2a cells were genetically modified by the use of pmuIL-12IRES1-hygro and/or pVkL/IL-15IRES1-neo plasmids as described previously (21). The characteristics of the clones secreting different cytokines are shown in Table 1. The selected clones displayed growth kinetics similar to that of Neuro2apc in vitro but were not tumorigenic when injected s.c. in syngeneic mice (data not shown). Neuro2a cells secreting IL-12 (Neuro2a/IL-12) and double-engineered cells (Neuro2a/IL-12/IL-15) were not tumorigenic also by i.v. injection, whereas Neuro2a cells secreting IL-15 (Neuro2a/IL-15) displayed a reduced tumorigenic potential (47% take) with respect to parental cells. Thus, in general, cytokine-gene transfer strongly decreased Neuro2a tumorigenicity in vivo, and s.c. injection of viable cytokine-engineered cells was selected for vaccination procedures in the following experiments.

Efficacy of Different Cytokine-Engineered Neuroblastoma Cells in Immunoprevention. Naive A/J mice were first vaccinated by s.c. injection of Neuro2a/IL-12, Neuro2a/IL-15, or Neuro2a/IL-12/IL-15 cells 30 days before i.v. injection of Neuro2apc. As shown in Fig. 1 A , all control animals died within 27 days, whereas Neuro2a/IL-12 and Neuro2a/IL-15 vaccination protected 45% and 28% of mice (P < 0.0001 and P = 0.0003), respectively. Vaccination with Neuro2a/IL-12/IL-15 cells protected only 28% of mice (P = 0.0009), showing that the simultaneous release of IL-12 and IL-15 by neuroblastoma cells does not improve the induction of systemic immunity (Fig. 1 A) in this tumor model, at variance with that observed in different models (17–21).



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  		Fig. 1 Immunopreventive effects of Neuro2a/IL-12, Neuro2a/IL-15, and Neuro2a/IL-12/IL-15 cells. A, survival of A/J mice that received Neuro2a/IL-12, Neuro2a/IL-15, and Neuro2a/IL-12/IL-15 cell vaccine (1 x 10 6 viable cells of each vaccine s.c. in a single site) 30 days before i.v. challenge with 1 x 10 6 Neuro2apc. B, survival of A/J mice that received Neuro2a/IL-12 vaccine 30 days and Neuro2a/IL-15 15 days before i.v. challenge with 1 x 10 6 Neuro2apc.

 
We then tested whether sequential injection of IL-12- and IL-15-engineered neuroblastoma cells could increase protection based on the hypothesis that IL-12 may activate a Th1 response (6) and IL-15 may subsequently act as a proliferative/survival factor for CD8+ effector/memory cells (25, 26). Sequential vaccination using Neuro2a/IL-12 cells at day –30 followed by Neuro2a/IL-15 at day –15 before Neuro2apc i.v. challenge protected 71% of mice (P = 0.0004 versus untreated mice; Fig. 1 B), showing a significant increase in survival rate with respect to single vaccinations (P = 0.145 for Neuro2a/IL-12 and P = 0.038 for Neuro2a/IL-15 and Neuro2a/IL-12/IL-15). Long-term surviving animals (>100 days from challenge) were completely free from disease as assessed by histologic and reverse transcription-PCR analysis for tyrosine hydroxylase in bone marrow (data not shown). S.c. injection of Neuro2a/IL-12 (day –30) followed by Neuro2apc (day –15) did not lead to significant changes in protection rate with respect to Neuro2a/IL-12 alone (data not shown).

The polarization of immune response triggered by the different vaccination protocols was investigated by IL-4 or IFN-{gamma} enzyme-linked immunospot assays on splenocytes isolated from the different groups of vaccinated mice on in vitro restimulation with Neuro2apc. No significant increases in cells responding to Neuro2apc by IL-4 secretion were observed in all groups of vaccinated mice with respect to naive mice (Fig. 2 A). On the opposite, splenocytes from all groups of vaccinated mice showed increased numbers of IFN-{gamma}-producing cells on Neuro2apc stimulation compared with splenocytes from naive mice (P < 0.01), which showed no response. In addition, the IFN-{gamma} response was higher (P < 0.05) in mice receiving the sequential vaccination protocol with Neuro2a/IL-12 and Neuro2a/IL-15 than in the other groups of vaccinated mice (Fig. 2 B). Depletion of CD8+ T cells from the splenocyte population of mice receiving the sequential vaccination protocol resulted in 85% inhibition of the IFN-{gamma} response, whereas CD4+ T-cell depletion had a limited effect on this response (Fig. 2 C). Altogether, these data indicated that a Th1/Tc1 polarization of the immune response was induced by all vaccination protocols used and that the sequential vaccination protocol seemed more efficient than the others in expanding a pool of Neuro2apc-specific CD8+ T cells. In addition, splenocytes from mice receiving the sequential vaccination protocol showed a slightly higher CTL activity against Neuro2apc cells than that of splenocytes from the other groups of vaccinated (P = NS) or unprimed mice (P < 0.05; data not shown).



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  		Fig. 2 Analysis of IL-4 and IFN-{gamma} production by splenocytes from mice receiving different cytokine-engineered vaccines. A, IL-4 production assessed by an enzyme-linked immunospot assay in the presence (black columns) or absence (white columns) of irradiated Neuro2apc cells. Splenocytes from the different groups (four mice per group) were restimulated at a 10:1 responder/target cell ratio for 36 hours. Spots were detected using a stereomicroscope. B, analysis of IFN-{gamma} production as above. C, splenocytes were treated with either anti-CD8 or anti-CD4 antibody and C' before the IFN-{gamma} enzyme-linked immunospot assay. Depletion was >90% as detected by immunofluorescence and fluorescence-activated cell sorting analysis.

 
Therapeutic Effect of Different Cytokine-Engineered Cells in Metastatic Syngeneic Neuroblastoma. First, Neuro2apc were injected i.v. in naive mice at day 0, and after 3 days, when micrometastatic nodules were already established (30), s.c. injection of cytokine-engineered cells was done. As shown in Fig. 3A, although all the mice finally developed metastatic disease and died, a significant increase in mean survival time (46 ± 27 versus 21 ± 3 days; P = 0.002) was observed in the group of mice receiving Neuro2a/IL-12 cells. Treatment at day +3 with either Neuro2a/IL-15 or Neuro2a/IL-12/IL-15 cells was devoid of any therapeutic activity (Fig. 3A).



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  		Fig. 3 Therapeutic effects of Neuro2a/IL-12, Neuro2a/IL-15, and Neuro2a/IL-12/IL-15 cells in Neuro2apc tumor-bearing mice. A, survival of A/J mice vaccinated s.c. with 1 x 106 Neuro2a/IL-12, Neuro2a/IL-15, and Neuro2a/IL-12/IL-15 cells 3 days after i.v. challenge with 1 x 106 Neuro2apc. B, survival of A/J mice vaccinated s.c. with Neuro2a/IL-12 at day +3 and with Neuro2a/IL-15 cells at day +13 after challenge with 1 x 10 6 Neuro2apc. C, reverse transcription-PCR analysis of tyrosine hydroxylase (TH) mRNA expression in bone marrow from a naive A/J mouse (lane 2) or from three untreated A/J mice bearing Neuro2apc micrometastases (lanes 3-5) and from six long-term surviving A/J mice, which were apparently cured from NB metastatic disease by sequential therapeutic vaccination (lanes 6-11). Lane 1, expression of tyrosine hydroxylase mRNA in Neuro2apc as control. Bottom, amplification products obtained with the G3PDH primers. Water was used as negative control (C-).

 
Next, the efficacy of sequential s.c. injection of Neuro2a/IL-12 at day +3 followed by Neuro2a/IL-15 cells at day +13 was tested. As shown in Fig. 3 B, a significant increase in the mean survival time was observed with respect to untreated mice (mean 66 ± 32 versus 21 ± 3 days; P < 0.0001). More importantly, 43% of mice showed a survival time exceeding 100 days, without signs of disease. No metastases were detected at autopsy by histopathologic analysis (data not shown), and only 3 of 6 mice displayed faint positivity for TH gene expression in the bone marrow by reverse transcription-PCR (Fig. 3 C). Rechallenge of another group of mice long-term (>100 days) surviving after sequential immunotherapy revealed that most (3 of 5) of these mice still showed protective immunity, thus confirming that a long-term protection is induced by the sequential vaccination protocol.

Splenocytes from Mice Responding to Therapeutic Vaccination Display CTL Activity and Secrete Cytokines. As shown in Fig. 4 A, mixed lymphocyte tumor cell–restimulated splenocytes from long-term surviving vaccinated mice showed cytotoxic activity against 51Cr-labeled Neuro2apc, whereas splenocytes from naive (Fig. 4 B) or untreated mice challenged with Neuro2apc (data not shown) showed no cytolytic activity. It is noteworthy that also unstimulated splenocytes from vaccinated mice lysed Neuro2apc (Fig. 4 A). The cytolytic activity was significantly inhibited by the addition of a blocking anti-CD3 antibody but not by an irrelevant immunoglobulin (Fig. 4 E), suggesting that recognition of target cells via the T-cell receptor/CD3 complex was required. In addition, splenocytes of long-term surviving mice showed no up-regulation of the nonspecific YAC target cell lysis with respect to splenocytes from naive mice (Fig. 4 C and D). Finally, splenocytes from vaccinated mice released higher amounts of IFN-{gamma}than splenocytes from naive mice, and IFN-{gamma} production could be further enhanced by Neuro2apc in vitro restimulation (Fig. 4 F).



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  		Fig. 4 Cytotoxic activity and cytokine production by splenocytes from rejecting mice. A, cytolytic activity of splenocytes from mice receiving therapeutic vaccination after 5 days co-culture with or without irradiated Neuro2apc. Percentage of lysis of 51Cr-labeled Neuro2apc in a 4-hour assay. SD did not exceed 5%. B, cytolytic activity of splenocytes from naive A/J mice against Neuro2apc evaluated as in A. C and D, cytolytic activity of the splenocytes from long-term survival (C) or naive mice (D) against YAC target cells. E, inhibition of the cytolytic activity of splenocytes from vaccinated mice in the presence of 1 µg/mL anti-CD3 or irrelevant mAb. F, IFN-{gamma} release by splenocytes from naive or vaccinated mice induced by restimulation with irradiated Neuro2apc as assessed by ELISA.

 
Role of Different Cell Subsets in Neuroblastoma Immunotherapy. To further investigate the mechanisms underlying the therapeutic effect of sequential vaccination, different groups of mice were repeatedly treated with anti-CD4, anti-CD8, or anti-NK cell depleting antibodies. As shown in Fig. 5 A , CD8+ cells were absolutely required for successful vaccination, because most mice treated with anti-CD8 antibody died in a timeframe similar to that of controls, which did not receive cytokine-engineered cells. In contrast, depletion of CD4+ and NK cells did not impair the protective effect of therapeutic vaccination (Fig. 5 A). These data unambiguously showed that, in this model, CD8+ CTLs were required for a successful immune response against neuroblastoma cells.



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  		Fig. 5 Role of CD8+ CTLs in therapeutic vaccination. A, effect of anti-CD8, anti-CD4, anti-asialo-GM1, or irrelevant antibody on the survival of A/J mice receiving therapeutic vaccination after challenge with 1 x 10 6 Neuro2apc. B, expression of MHC class I molecules on NB cells isolated from a representative metastasis (Neuro2a ex vivo) developed in untreated mice and from three metastases developed in mice receiving therapeutic vaccination (M1, M2, and M3) as assessed by indirect immunofluorescence and cytofluorimetric analysis. All Neuro2a ex vivo were tested after 1 week of in vitro re-culture when they were >90% pure as assessed by morphologic examination. Numbers in parentheses, percentage of MHC class I–negative cells.

 
Several pieces of evidences indicate that loss or down-regulation of MHC class I expression by tumor cells represents a common tumor-escape mechanism from CTL-mediated immune control (31–34). Therefore, we analyzed the MHC class I expression in neuroblastoma cells recovered from tumors developed in mice that had not been cured by the therapeutic sequential vaccination and from tumors developed in untreated mice. As shown in Fig. 5 B, a relevant percentage of Neuro2a cells isolated from metastases of vaccinated mice showed a decreased expression of MHC class I, further suggesting an escape of some tumor cells from CD8+ CTLs in vivo.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we showed that cytokine-engineered Neuro2a cells display reduced to absent tumorigenic potential in syngeneic mice and can be used as efficient cellular vaccines in both preventive and therapeutic settings. In particular, the sequential administration of Neuro2a/IL-12 cells for primary immunization followed by Neuro2a/IL-15 for boosting allowed to achieve the highest rates of protection, reaching 71% in the immunopreventive and, more importantly, 43% in the immunotherapeutic setting. Previous studies in neuroblastoma models showed preventive or therapeutic efficacy of neuroblastoma murine cells engineered with IL-12 or other cytokines, but IL-15 was never tested in this experimental model. In addition, in most studies (35–38), the efficacy of IL-12 was evaluated in mice injected s.c. with neuroblastoma parental cells, a model that poorly mimics the human neuroblastoma disease.

The therapeutic effect reported herein was obtained in a very aggressive model of neuroblastoma established micrometastases, resembling human stage 4 disease (29). Moreover, vaccine therapy was started 3 days after i.v. Neuro2apc challenge, when micrometastastes were histologically detectable (30) and allowed a quite short therapeutic window; untreated mice in fact died within 27 days from challenge. In addition, in the same experimental setting, treatment with Neuro2a/IL-15 cells did not show any therapeutic effect, whereas Neuro2a/IL-12 partially increased the mean survival time. More intriguingly, Neuro2a cells coexpressing IL-12 and IL-15 failed to induce antitumor effects, thus proving to be less efficient than Neuro2a/IL-12 cells in spite of the secretion of similar levels of IL-12. This finding differs from previous reports in other tumor models, where coadministration of suboptimal amounts of recombinant IL-12 and IL-15 (17–19) or coexpression of these two cytokines (20, 21) produced synergistic antitumor effects. However, no synergistic effects of IL-12 and IL-15 coexpressed by mammary adenocarcinoma cells were observed in the immunocompetent syngeneic TS/A tumor model, 6 whereas a strong synergistic effect was observed in syngeneic IFN-{gamma}–/– mice (20). Previous reports indicated that IL-12 and IL-15 induce a strong costimulatory effect on the production of several cytokines, including IFN-{gamma} (39), and that IFN-{gamma}overproduction may result in the apoptotic cell death of T or NK cells (40). IFN-{gamma} is a cytokine endowed with strong antitumor functions related both to the induction of an antiangiogenic cascade and to direct effects on tumor cells, including sensitization to apoptosis (41) and inhibition of proliferation (42) of neuroblastoma cells. However, IFN-{gamma} has been more recently identified as a mediator of the activation-induced cell death of T lymphocytes (40), which represents a negative feedback mechanism of control of exaggerated immune responses (43). Thus, the simultaneous paracrine coexpression of IL-12 and IL-15 may result in local hyperinduction of IFN-{gamma}, leading to activation-induced cell death of locally recruited effector cells. It is conceivable that these phenomena would prevent the induction of an optimal immune response. Our present results indicated that the inefficacy of IL-12 and IL-15 coexpressing tumor cells can be circumvented when Neuro2a/IL-12 and Neuro2a/IL-15 cells are given sequentially. This schedule may allow a Th1 priming effect by IL-12 (6) followed by an IL-15 stimulus to CTL clonal expansion and to memory T-cell induction and/or maintenance (23). Indeed, the sequential vaccination protocol resulted in the induction of a Th1/Tc1 polarized response, of an enhanced CD8+ T-cell response, and the therapeutic efficacy of this treatment was strictly dependent on CD8+ T cells as indicated by in vivo antibody depletion experiments. In addition, splenocytes from mice showing long-term survival after immunotherapy showed CTL activity against Neuro2a cells. Although CTL activity was higher after mixed lymphocyte tumor cell restimulation of splenocytes, culture without antigen restimulation was sufficient to induce this activity, suggesting the in vivo presence of effector CTLs or of effector/memory CTLs displaying lytic functions in long-term surviving mice. Moreover, the finding that tumor cells isolated from late metastases of long-term surviving mice showed decreased MHC class I expression is also suggestive of an in vivo selective pressure by CTLs as reported recently in a similar murine neuroblastoma model (44) and in melanoma patients undergoing immunotherapeutic protocols (45).

Attempts to identify the possible antigen(s) recognized by the CTLs induced following Neuro2a cellular vaccines are in progress. In several other murine tumor models (46–48), the gp70env endogenous retroviral protein behaves as an immunodominant antigen and may mask responses to less immunogenic molecules. Preliminary results indicated that, although Neuro2a cells express gp70env mRNA, gp70env is not an immunodominant antigen in this model (data not shown).

Based on antibody depletion experiments, CD4+ and anti-asialo-GM-1+ NK cells did not seem to be strictly required for the therapeutic efficacy of the vaccines; however, a possible involvement of these cells cannot be excluded. It is likely that the proliferative/survival signal provided by tumor-released IL-15 to activated CD8+ cells may override the requirement of T-helper factors, particularly in advanced stages of the response. In addition, in view of the down-regulation of MHC class I expression observed in late metastasis, it cannot be excluded that NK cells contribute to tumor control.

In conclusion, our data indicate that the combined use of tumor cell vaccines made with IL-12 and IL-15 gene-modified cells represent a suitable approach to activate strong CD8+ T-cell responses, allowing the achievement of therapeutic effects in an aggressive metastatic neuroblastoma model. Finally, the sequential use of IL-12 and IL-15, rather than their simultaneous administration, is an efficient adjuvant combination for T-cell-based immunogene therapy.


   ACKNOWLEDGMENTS
 
We thank C. Bernardini for excellent secretarial assistance.


   FOOTNOTES
 
Grant support: Associazione Italiana per la Ricerca sul Cancro, Fondazione Italiana per la lotta al Neuroblastoma and Compagnia San Paolo (S. Ferrini), Ministero della Salute (M.V. Corrias), and Fondazione Italiana per la Lotta al Neuroblastoma fellowships (M. Croce and B. Carlini).

Note: M.V. Corrias and S. Ferrini equally contributed to this work.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

6 Comes et al., unpublished observations. Back

Received 4/28/04; revised 9/30/04; accepted 10/13/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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