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Immunology |
Departments of Surgery [T. T., A. G., W. J. S.] and Molecular Genetics and Biochemistry [P. D. R., W. J. S.], University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5-transfected DCs or nontransduced DCs significantly inhibited the growth of established tumors but did not lead to complete regression of established tumors. Importantly, immunization with antigen-loaded AdIL18DC resulted in tumor rejection or further suppression of tumor growth when compared with controls. The repertoire of naturally presented tumor peptides recognized by splenocytes (as deduced in IFN-
ELISA assays) from AdIL18DC-treated animals was far more diverse and of greater magnitude than that of all other groups, in association with improved therapeutic outcome. These results support the ability of IL-18 gene transfer to enhance the capacity of DCs to drive broadly reactive Th1-type therapeutic immunity prompted by single peptide epitope-based vaccines (i.e., epitope spreading). |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IL-18 was originally identified as a monokine with important immunoregulatory functions, including the ability to induce high levels of IFN- secretion from both NK and T cells (i.e., IFN--inducing factor; Ref. 15
). IL-18 is a Mr 18,300 member of the IL-1 family of proinflammatory cytokines, produced by activated macrophages and DCs, that appears to play an important role in driving Th1-dominated immune responses (16
, 17)
.
Recently, the role of IL-18 as a biological "adjuvant" has been evaluated in murine tumor models. Systemic administration of rIL-18 induces significant antitumor effects in multiple murine tumor models (18 , 19) . However, rIL-18 administration has prompted severe "septic shock-like" toxicities, particularly when combined with rIL-12, that may ultimately prevent the widespread clinical application of this recombinant protein (20) . To overcome such systemic toxicities, we examined the effectiveness of therapeutic immunization with genetically transduced DCs to provide paracrine secretion of IL-18 in the microenvironment of evolving antitumor T-cell activation. We demonstrate the novel finding that IL-18 gene-modified DCs not only potentiate the antitumor effects of single synthetic peptide-based immunizations in tumor-bearing mice, but they also prompt the evolution of a larger and more diverse "therapeutic" repertoire of specific antitumor CTLs in situ.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines and Culture.
CMS4 sarcomas (H-2d) express mutated p53 and present the wild-type p53232–240 epitope recognized by H-2Kd-restricted CTLs (21
, 22)
. YAC-1, a NK cell-sensitive cell line, was a generous gift of Dr. William H. Chambers (University of Pittsburgh). These cell lines were maintained in complete media, as described previously (22)
.
Generation of DCs in Vitro from Bone Marrow.
DCs were generated from BALB/c bone marrow in 7-day cultures as described previously (23)
and isolated at the interface of 14.5% (w/v) metrizamide (Sigma, St. Louis, MO) in CM discontinuous gradients by centrifugation. DCs expressed CD11b, CD11c, CD40, CD54, CD80, CD86, and MHC class I and class II antigens (data not shown).
Viral Vectors.
The mock adenoviral vector Ad
5 was used as a control virus, as described previously (24)
. The adenovirus encoding mouse IL-18 gene under a cytomegalovirus promoter (AdmGM/IL18) and E1- and E3-deleted adenoviral vectors were constructed through Cre-lox recombination with reagents generously provided by Dr. Stephen Hardy (Somatix, Alameda, CA; Ref. 25
). The cDNA encoding the mature mouse IL-18 fused with a mouse GM-CSF leader sequence was obtained by PCR amplification using a 60-bp 5' primer corresponding to mouse GM-CSF leader sequence (40 bp) and 20 bp of annealing to the mature mouse IL-18 coding region. The digested PCR fragment containing the mouse granulocyte/macrophage/IL-18 cDNA was subsequently inserted into shuttle vector pAdlox and sequenced. Recombinant adenovirus was generated by cotransfection of SfiI-digested pAdlox-mGM/IL18 andy5 helper virus DNA into the adenoviral packaging cell line CRE8 that expresses Cre recombinase. Recombinant adenoviruses were amplified in CRE8 cells, purified by CsCl density gradient centrifugation, and then dialyzed. Titers of viral particles were determined by optical densitometry, and viral particles were then stored at -80°C until use.
Mouse IL-18 and IL-12 Production from Adenoviral-transduced DCs.
Five million DCs were transfected with recombinant adenoviral vector encoding mouse IL-18 (AdIL18) or mock vector (Ad5), as reported previously (24)
. After 48 h, adenoviral transfected DCs were harvested and analyzed. Culture supernatants were also collected for measurement of mouse IL-18 and mouse IL-12 production using specific mouse IL-18 and mouse IL-12 ELISA kits (BD PharMingen, San Diego, CA), with lower levels of detection of 31.3 and 62.5 pg/ml, respectively.
MLR.
Adenoviral transfectants and nontransduced DCs were irradiated (30 Gy) using a 60Co gamma irradiator (Nordion International Inc., Ontario, Canada), washed with RPMI 1640 (Life Technologies, Inc.), and seeded in triplicate (10–2.5 x 103 cells/well) in round-bottomed 96-well plates for use as stimulator cells. Allogeneic (C57/BL6) splenic responder CD3+ T cells (2 x 104) were added to DCs (at the indicated T cell:DC ratios) in a total volume of 200 µl of CM and cultured for 96 h. The wells were then pulsed with 1 µCi of [3H]thymidine (New England Nuclear, Boston, MA) for the last 18 h of incubation, and cells were harvested onto glass fiber filters (Wallac, Gaithersburg, MD). [3H]Thymidine incorporation was quantified using a beta plate liquid scintillation counter (Wallac), with results expressed as the mean cpm ± SE (SD) of three independent experiments.
Flow Cytometry.
For phenotypic analysis of adenoviral transfected DCs, phycoerythrin- or FITC-conjugated monoclonal antibodies against mouse cell surface molecules [CD11b, CD11c, CD40, CD54, CD80, CD86, H-2Kd, and I-Ad (all from BD PharMingen)] and appropriate isotype controls were used, and analysis was performed using a FACscan (Becton Dickinson, San Jose, CA) flow cytometer.
Animal Experiments.
BALB/c mice were injected s.c. with 3 x 105 CMS4 cells in the right flank on day 0. On day 7, when tumor size reached approximately 20–30 mm2, BALB/c mice were treated s.c. (on the same flank of tumor) with immunization with 1 x 106 nontransduced or adenoviral transduced DCs prepulsed with the mouse p53232–240 peptide in a total volume of 100 µl of PBS as described previously (21)
, and 1 week later, 1 x 106 nontransduced or adenoviral transduced DCs were injected intratumorally in a total volume of 100 µl of PBS. Tumor size was assessed every 3 or 4 days and recorded in mm2 by determining the product of the largest perpendicular diameters measured by vernier calipers. Data are reported as the average tumor area ± SD. To assess the impact of systemic immunity from vaccination, we examined the growth of contralateral untreated tumors. For the latter models, BALB/c mice were injected s.c. with 3 x 105 CMS4 cells in both flanks on day 0. On day 7, BALB/c mice were treated with vaccines as noted above. After 7 additional days, 1 x 106 adenoviral transduced DCs were injected in the tumor on the right flank, and both tumors were measured every 3 or 4 days.
Cytolytic Assay and IFN- ELISA.
Splenocytes were harvested from 2 mice/group 7 days after intratumoral injection with adenoviral transduced DCs (i.e., day 21 after tumor inoculation). Responder cells (3 x 106 cells/well) were restimulated in vitro with 3 x 105 irradiated (10,000 rads) CMS4 cells in the presence of 30 IU/ml recombinant human IL-2 (Chiron Corp., Emeryville, CA) for 5 days in 24-well culture plates. The supernatants were collected and assessed in an ELISA for mouse IFN- production (BD PharMingen). Lymphocytes were harvested after 5 days of in vitro restimulation and subjected to 5-h 51Cr release assays against the CMS4 and YAC-1 targets, as described previously (26)
. Assays were performed in triplicate wells, with spontaneous release of all assays never exceeding 25% of the maximum release.
CD8+ T-Cell Response against Eluted Naturally Processed Peptides Derived from CMS4 Cells.
Peptides were extracted from CMS4 cells as described previously (27)
and separated on reverse-phase HPLC. Individual HPLC fractions were lyophilized to remove organic solvent and then reconstituted in 200 µl of PBS and stored at -20°C until use. CD8+ T cells were isolated from the spleen cells of immunized mice by using magnetic beads (MACS; Miltenyi Biotec, Aubum, CA) and then cocultured (1 x 105 cells/well) with syngeneic DCs (2 x 104 cells/well) pulsed with HPLC-fractionated peptides in 96-well tissue culture plates. After 48-h incubations, culture supernatants were collected and analyzed for IFN- release using a cytokine-specific ELISA kit (BD PharMingen).
Statistical Analysis.
Statistical significance of differences between the groups was determined by applying Student’s t test or two-sample t test with Welch correction after each group had been tested for equal variance and Fisher’s exact probability test. Statistical significance of the differences in more than three groups was determined by applying one-way ANOVA. We defined statistical significance as P < 0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5-transduced DCs (Ad5DC) and nontransduced DCs (DCs) did not secrete detectable levels of mIL-18 (i.e., <31.3 pg/5 x 106 cells/48 h). Using flow cytometric methods, we determined that expression of MHC class I, MHC class II, CD80, CD86, and CD40 molecules on AdIL18DC was significantly elevated when compared with either Ad5 control virus-infected DCs and nontransduced DCs (Table 1)
, with these controls yielding indistinguishable results. When used as stimulators in MLRs, IL-18-transduced DCs exhibited significantly higher T-cell proliferation than Ad5-infected DCs or nontransduced DCs (Fig. 1)
. Because rIL-18 and recombinant IFN- stimulate increased IL-12p70 production from DCs, which is critical to the induction of Th1-type immunity, the supernatants of transduced DCs were also tested for production of IL-12 heterodimers. IL-12p70 production from IL-18-transduced DCs was significantly higher than that from either Ad5-infected DCs or nontransduced DCs (127 pg/5 x 106 cells/48 h versus <62.5 pg/5 x 106 cells/48 h for either control).
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5- or mIL-18-transduced) DCs pulsed with the p53232–240 peptide. In some groups, after an additional 7 days, tumors were injected with 1 x 106 nontransduced or adenoviral transduced DCs or PBS to direct primed immune effectors into the tumor to theoretically promote cross-presentation and "epitope-spreading." As shown in Fig. 2A
, tumor rejection was observed in three of six mice treated with AdIL18DC, and the growth of CMS4 tumors in the remaining three mice was significantly inhibited compared with that seen in mice treated with the other protocols (P < 0.05 at 14, 17, 21, and 24 days versus PBS; P < 0.05 at 14, 17, 21, 24, and 28 days versus Ad5DC or DC). Although immunization with Ad5-infected DCs or nontransduced DCs did not lead to complete regression of established tumors, tumor growth was significantly inhibited compared with that seen in mice treated with PBS (P < 0.05 at 17, 21, and 24 days versus PBS). As shown in Fig. 2B
, the tumor growth of CMS4 cells in mice treated intratumorally with AdIL18DC was significantly inhibited compared with that seen in mice treated intratumorally with Ad5-infected DCs, nontransduced DCs, or PBS (P < 0.05 at 17, 21, 24, and 28 days). These results demonstrate that IL-18 gene transfer enhances the therapeutic effects of DC-based immunization (provided 1 week earlier) against established CMS4 tumors or, alternatively, that prevaccination with a single tumor epitope enhanced the efficacy of intratumoral IL-18 gene therapy.
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5-infected DCs or PBS. After 7 days, all mice were treated with intratumoral injection of DC-AdmIL18. As shown in Fig. 2C
, the tumor growth in mice treated with p53 peptide-pulsed AdIL18DC was significantly inhibited compared with that seen in mice treated with any other combined therapy (P < 0.05 at 14, 17, 21, 24, and 28 days). These results demonstrate that p53 peptide-pulsed DCs have therapeutic potential against CMS4 tumors, particularly if AdIL18DC is used as a vaccine, and that subsequent intratumoral delivery of AdIL18DC is provided in a combinational gene therapy.
Induction of Cytolytic Activity against CMS4 Cells and IFN- Production.
We next examined whether transfection of DCs enhances their ability to elicit specific anti-CMS4 CTL generation after combinational therapy (as evaluated in Fig. 2
). Spleen cells were obtained from mice 14 days after intratumoral injections and cocultured with irradiated CMS4 cells for 5 days. As shown in Fig. 3A
, splenocytes from mice treated with the Ad5-infected DC regimen (i.e., p53 peptide-pulsed Ad5DC vaccine + Ad5DC intratumoral injection) or the nontransduced DC regimen displayed low-level cytolytic activity against CMS4 cells, whereas CTLs from mice treated with PBS were nonreactive. Importantly, splenocytes from mice treated with the AdIL18DC regimen displayed significantly stronger cytolytic activity versus either the Ad5DC or nontransduced DC regimens. As shown in Fig. 3B
, no cytolytic activity was observed against the NK/LAK target YAC-1. These results demonstrate that the combined AdIL18DC vaccination/intratumoral injection therapy was superior in its ability to stimulate systemic CTL activity against a resident tumor in situ. Splenocytes obtained from mice treated with the AdIL18DC regimen also produced substantially elevated amounts of the Th1-associated cytokine IFN- (>10,000 pg/ml) as compared with effector cells obtained from mice treated with other DC regimens (Ad5DC = 2,980 pg/ml, DC = 1,189 pg/ml), which in turn exceeded production from mice injected with PBS only (68 pg/ml; Fig. 3C
).
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, the uninjected tumors in mice treated with the AdIL18DC regimen were significantly smaller compared with those in mice treated with either PBS (P < 0.05 at day 21, 24, and 28) or the Ad5DC regimen.
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production by CD8+ T cells was measured by ELISA after coculture with autologous DCs pulsed with naturally presented, HPLC-separated peptides isolated from CMS4 cells. As shown in Fig. 5
, CD8+ T cells from AdIL18DC regimen-treated mice were observed against a variety of peptides, including fractions coeluting with the p53232–240 peptide, used to immunize these mice. CD8+ T cells from animals treated with the Ad5DC or nontransduced DC regimens also reacted against peptides in some HPLC fractions, but the number of reactive fractions and the magnitude of the T-cell responses in these groups were far lower than those promoted by AdIL18DC. CD8+ T cells from PBS-treated mice did not produce detectable levels of IFN- against any peptides. These results demonstrate that the DC-based combined therapies (based on a single synthetic epitope) can induce spreading in the CD8+ T-cell responses to CMS4-derived peptides, with the greatest degree of "epitope spreading" noted for therapies using DC-IL18. Notably, these expanded CTL repertoires were associated with increased therapeutic benefit.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B via recruitment of MyD88 (28)
, which regulates the expression of these (MHC and costimulatory) molecules. Thus, IL-18-induced phenotypic changes could be due to the direct effects of IL-18 and/or indirect effects, such as those promoted via IFN- production from IL-18-activated T cells or NK cells, that represent minor contaminants (<10%) in our DC preparations. MLR results demonstrate that the allostimulatory capacity of IL-18-transduced DCs was significantly greater than that of control DCs, consistent with the observed changes in DC costimulatory and MHC marker phenotype and the enhanced therapeutic efficacy that we observed in the CMS4 model. In the current study, mice bearing established CMS4 tumors were treated with combinational immunotherapy incorporating p53232–240 peptide-pulsed DC vaccines 7 days after tumor inoculation, followed by intratumor injection of (non-antigen-loaded) DCs 1 week later. We theorized that the first vaccination of tumor-bearing mice results in increased frequencies of p53 peptide-specific CTLs that circulate and, after infiltrating tumors, result in lesional destruction and tumor antigen acquisition by infiltrating (or injected) DCs in situ, thereby promoting subsequent "cross-priming" of tumor-reactive T cells in the tumor-draining lymph nodes. These cross-primed T cells would exhibit an expanded repertoire of antitumor specificities capable of mediating more effective tumor clearance and greater immune protection against tumor cells expressing heterogeneous levels of individual antigens. Such cross-priming would be theoretically enhanced by intratumor injection of DCs (serving as cross-presenters), particularly if these cells were engineered to express a proinflammatory cytokine (such as IL-18) to recruit immune cells and promote Th1-type immunity.
Our results demonstrate that established tumor growth in mice immunized with peptide-pulsed DCs was significantly inhibited compared with that of tumors in mice immunized with non-peptide-pulsed DCs or PBS (in groups receiving a common intratumoral treatment), suggesting that the immunization phase of the combinational therapy plays an essential role in "priming" for subsequently broadened antitumor immune responses. Because tumor growth in mice subsequently treated with intratumoral injections of AdIL18DC on day 14 was further inhibited compared with controls, it is likely that intratumoral injection of AdIL18DC boosts antitumor immunity (which was promoted initially by the primary p53 peptide-pulsed DC immunizations) most effectively. Taken together, these observations suggest that both the initial immunization and the subsequent intratumoral injection of DCs play important roles in the induction and direction of therapeutic antitumor immunity in tumor-bearing mice, with intratumoral IL-18-transduced DCs optimally enhancing these effects in the current study. Major issues that have not been addressed by this study, but which we are currently evaluating, include the requirement for IL-18 gene therapy delivery via adenovirus-infected DCs (versus infected fibroblasts or direct injection of AdIL18 virus ± control DCs) for therapeutic efficacy and the IL-18 dose dependency of therapeutic efficacy.
Importantly, combinational vaccination using IL-18-transduced DCs also exhibited therapeutic effects against nontreated contralateral tumors, supporting the ability of this vaccination protocol to induce systemic antitumor immunity. These results suggest that immunization of cancer-bearing animals with IL-18 gene-transduced DCs may represent a promising approach to suppress tumor growth and ultimately regress disseminated lesions in cancer patients.
We have shown that immunization of tumor-bearing mice with p53 peptide-pulsed DCs promotes CMS4-specific CTL cytolytic activity and IFN- production in the spleens of treated animals, particularly if IL-18-transduced DCs were applied in situ. Zitvogel et al. (29)
reported that the antitumor effects of DC-based vaccination were dependent on production of Th1-associated cytokines such as IFN-, tumor necrosis factor 
, and IL-12. Therefore, enhanced IFN- production resulting from IL-18 paracrine delivery via engineered DCs (promoted by either IL-18 or up-regulated IL-12) may also play an important role in the increased antitumor activity in vivo.
Previous reports have demonstrated that both CTLs and NK cells play important roles in the antitumor effects induced by systemic administration of IL-18 in murine tumor models (18 , 19) . Osaki et al. (24) reported that direct injection of an IL-18 adenovirus into tumor combined with systemic administration of IL-12 exerted antitumor effects mediated mainly by NK cells and partially by both CD8+ and CD4+ lymphocytes. We initially expected that the Th1 cytokine IL-18 produced by transduced DCs might also enhance the antitumor activities of both CTLs and NK cells induced by the vaccine. Our results indicate that specific CTL, but not NK cell, activity is associated with the improved efficacy associated with our IL-18-based combinational therapy in the CMS4 model. This finding is consistent with our in vitro data supporting IL-18-mediated changes in DC phenotype and function relevant for T-cell activation. These results do not discount the potential important role that NK cells may play in licensing Th1-type immunity (30) but suggest that their direct antitumor effects in this model may be limited.
A number of recent examples suggest that the induction of (at least) limited autoimmunity and "epitope spreading" may drive the most effective therapeutic immune responses to cancer (31 , 32) . Indeed, in the therapy of established mice and human melanoma, the development of autoimmune vitiligo (destruction of normal melanocytes in the skin) is associated with better objective clinical responses (32 , 33) . Our results demonstrate that epitope spreading can also be best observed in mice that have been effectively treated with combinational therapy consisting of specific vaccination and subsequent intratumoral delivery of IL-18-transduced DCs. It will clearly be of great interest to prospectively evaluate the comparative effects of IL-18-transduced DCs loaded with diverse forms of tumor antigens (apoptotic bodies, lysate, conjugates, and peptides) to stimulate the greatest diversity in tumor-specific T-cell reactivity and discern the coordinate therapeutic benefits of these approaches.
Despite recent progress and early success reported for DC-based cancer immunotherapies, there is significant room for improvement in these regimens. To that end, we have demonstrated several novel findings in the current report, namely that (a) IL-18 transfection of DCs enhances their immunostimulatory phenotype/function, (b) IL-18 gene therapy (using DCs) provides superior efficacy when applied in combinational approaches as compared with vaccines only or intratumoral delivery applications only, and (c) the therapeutic efficacy of these combinational therapies is associated with epitope spreading in the antitumor CD8+ T-cell repertoire that can be effectively primed using single peptide-based vaccines. Given current Food and Drug Administration concerns regarding the direct injection of recombinant adenoviruses into patients and the limited efficacy observed for single peptide-based cancer vaccines (34) , these findings may provide the basis for safer, more effective cancer therapies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants CA 63350 (to W. J. S.) and CA 68067 (to A. G., P. D. R., and W. J. S.). 
2 To whom requests for reprints should be addressed, at Department of Surgery, University of Pittsburgh School of Medicine, W1555 Biomedical Sciences Tower, 200 Lothrop Street, Pittsburgh, PA 15261. Phone: (412) 624-6453; Fax: (412) 624-1172; E-mail: storkuswj{at}msx.upmc.edu
3 The abbreviations used are: DC, dendritic cell; IL, interleukin; rIL, recombinant interleukin; GM-CSF, granulocyte/macrophage colony-stimulating factor; NK, natural killer; MLR, mixed leukocyte reaction; HPLC, high-performance liquid chromatography; mIL, murine interleukin; CM, complete media; LAK, lymphokine activated killer cells.
Received 5/14/02. Accepted 8/15/02.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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inducing factor/IL-18 administration mediates IFN-- and IL-12-independent antitumor effect. J. Immunol., 160: 1742-1749, 1998.-dependent and -independent mechanisms in adverse effects caused by concomitant administration of IL-18 and IL-12. J. Immunol., 164: 3330-3336, 2000. inducing factor, interleukin-18 (IL-18). Gene Ther., 6: 808-815, 1999.[Medline]
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), Ad
), and nontransduced DCs (DC, 
) as described in "Materials and Methods." The data are shown as the mean ± SD. Similar results were obtained in three independent experiments. *, P < 0.05 versus Ad
), or nontransduced DCs (
and 
). C, vaccine-dependent effects on the efficacy of combined therapies. BALB/c mice bearing established CMS4 tumors were immunized with 1 x 106 p53232–240 peptide-pulsed DCs (as indicated), and after an additional 7 days, tumors were injected with 1 x 106 non-peptide-pulsed AdIL18DC. In all cases, the fraction of mice bearing a tumor in each treatment group at 30 days is indicated in parentheses. Tumor size was expressed as the mean tumor size of only those mice bearing tumors. Each data point represents the mean tumor size ± SD. These experiments were performed twice, and essentially identical results were obtained.
