Potent Vaccine Therapy with Dendritic Cells Genetically Modified by ...

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doi:10.1016/j.ymthe.2005.09.021

Potent Vaccine Therapy with Dendritic Cells Genetically Modified by the Gene-Silencing-Resistant Retroviral Vector GCDNsap Tsukasa Nabekura,1 Makoto Otsu,1 Toshiro Nagasawa,1 Hiromitsu Nakauchi,2 and Masafumi Onodera1,* 1

Division of Clinical and Experimental Hematology, Major of Advanced Biomedical Applications, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan 2 Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Science, The University of Tokyo, 4-6-1 Shiroganedai, Minatoku, Tokyo 108-8639, Japan *To whom correspondence and reprint requests should be addressed. Fax: +81 29 853 7499. E-mail: [email protected].

Available online 28 November 2005

Dendritic cells (DCs) genetically modified to express tumor-associated antigens (TAAs) would be promising tools in cancer immunotherapy. However, the use of retroviral vectors for such modifications is still a challenge because of low transduction efficiency and gene silencing in DCs. We have established an efficient method to prepare such DCs by in vitro differentiation of hematopoietic progenitor cells transduced with chicken ovalbumin (OVA) cDNA via the genesilencing-resistant retroviral vector GCDNsap packaged in vesicular stomatitis virus G protein. When c-KIT+/lineage cells were transduced with OVA followed by expansion and differentiation, more than 90% of mature DCs expressed the transgene. Mice inoculated with those cells completely rejected the OVA-expressing tumor E.G7-OVA, and the anti-tumor effects were stronger than those observed in mice inoculated with the same number of OVA peptide-pulsed DCs. The mice harbored more cytotoxic T lymphocytes (CTLs) against E.G7-OVA and produced antibody against OVA, suggesting the generation of multiple CTLs recognizing different OVA epitopes and OVA-specific CD4+ T cells. Successive inoculations of the transduced DCs in a therapeutic setting eradicated preexisting E.G7-OVA and prevented the progression of retransplanted tumors. Thus, this vaccine therapy may represent a potent immunotherapeutic approach for various malignant tumors that express suitable TAAs. Key Words: dendritic cell, retrovirus, gene transfer, cancer vaccine, cancer immunotherapy, GCDNsap, OVA, tumor immunology, gene silencing

INTRODUCTION Dendritic cells (DCs) are the most powerful professional antigen-presenting cells [1–3]. Because they express various immunostimulatory molecules on their surface, these cells are capable of priming both resting and naRve T lymphocytes without additional exogenous adjuvants and of eliciting antigen-specific immune responses [1– 5]. In addition to these unique characteristics, the fact that DCs can be induced with cytokines in vitro either from hematopoietic stem/progenitor cells (HSCs) in marrow or from monocytes in peripheral blood allows in vitro manipulation of the DCs to induce the presentation of tumor-associated antigen (TAA)-derived peptides through their MHC (major histocompatibility complex) molecules and facilitates their use as an attractive

MOLECULAR THERAPY Vol. 13, No. 2, February 2006 Copyright C The American Society of Gene Therapy 1525-0016/$30.00

immunotherapeutic tool in the treatment of malignant tumors [6–8]. To date, a variety of methods to prepare such DCs have been devised in experimental studies and applied in clinical trials, including exposure of DCs to synthetic peptides [9–11] or tumor lysates [10,12,13], fusion with tumor cells [14–16], and transfection with RNA [12,17,18] or DNA [19–21] encoding TAAs. However, there are still some obstacles that prevent the effective use of these DCs in clinical applications [8,22], e.g., the preparation of substantial tumor masses or samples from inaccessible tumors is quite difficult in the clinical setting using tumor lysates or tumors per se. An alternative is to load TAA-derived epitope peptides on the MHC molecules of DCs (so-called bpeptide-pulsed DC vaccineQ) [23]. However, few clinical trials using peptide-pulsed DCs

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have proved effective [24,25], partly because the few available peptides restricted to the MHC class II molecules cannot induce tumor-specific CD4+ T lymphocytes. Although DCs transduced with full-length TAA cDNAs with viral vectors for presentation of tumor epitopes through the MHC have been tested, low transduction efficiency, cytotoxicity or immunogenicity of viral vectors, and short-term presentation of TAAs are still challenges [26]. To address these issues, another promising approach, i.e., the use of DCs differentiated from HSCs genetically modified with retroviral vectors, has been proposed [27– 29]. Such DCs have several advantages over the conventional methods described above, e.g., stable expression of TAAs by DCs leading to appropriate processing of TAAderived peptides for both MHC classes I and II [28,30], the possibility of establishment of TAA-specific CD4+ helper T cells and production of TAA-specific antibodies [27,30,31], and the possibility of generating a substantial number of the TAA-transduced DCs by in vitro expansion of the transduced HSCs with cytokines [26,28]. Despite these advantages, the anti-tumor effects observed to date have been far from satisfactory because of the low transduction efficiency of HSC-derived DCs with current retroviral technologies [22]. In addition, down regulation of the transgene expression (gene silencing) in mature DCs remains a critical problem for efficient priming and activation of cytotoxic T lymphocytes (CTLs) against tumors [29,32]. We have developed a retrovirus-mediated gene transfer system with the gene-silencing-resistant retroviral vector GCDNsap packaged using vesicular stomatitis virus G protein (VSV-G). The system has allowed continued expression of the transgene by HSCs [33,34], neural stem cells [35], and hepatic stem cells [36] in addition to high transduction efficiency. In this study, we analyzed the usefulness of DCs differentiated from HSCs genetically modified with this vector system as an immunotherapeutic agent both in vitro and in vivo.

RESULTS Characterization of Chicken Ovalbumin (OVA)-Transduced DCs Both the structure of the GCDNsap vector encoding the full-length OVA cDNA (Fig. 1A) and the method used to produce the OVA-expressing recombinant retroviruses packaged in the VSV-G envelope (GCDN/OVA) are described under Materials and Methods. The titer of the concentrated virus was approximately 9 106 infectious units/ml, as measured on Jurkat cells. We stimulated murine c-KIT+/lineage-negative cells (KL cells) obtained from C57BL/6 mice on Day 0 with mouse interleukin (IL)3, mouse IL-6, mouse stem cell factor (SCF), human thrombopoietin, and human Flt3 ligand and transduced them with the OVA cDNA on Day 1 and Day 2 by

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exposure to 50 Al of virus-containing culture supernatant. After expansion for an additional 5 days, we transferred the cells to cultures containing mouse IL-4 and mouse granulocyte/macrophage colony-stimulating factor (GMCSF) (on Day 8) and collected them on Day 14 for use in subsequent experiments. At that point, most of the cells were morphologically mature DCs with high expression of CD11c, CD40, CD86, and MHC class II on their surface, and the transduction efficiency, as estimated by enhanced green fluorescent protein (EGFP) expression, was over 90% (Figs. 1B and 1C). The number of OVAtransduced DCs obtained was approximately 5 106 cells, which is 20 times more than the number of DCs present in freshly harvested bone marrow. We confirmed OVA expression in the transduced DCs by Western blot analysis (data not shown). To assess whether OVA produced in the DCs was properly processed to peptides and presented by the MHC class I molecules to yield OVA-specifically activated T cells, we cocultured the transduced DCs with B3Z, a unique T cell hybridoma in which lacZ activity is specifically induced in response to the OVA peptide (SIINFEKL)/MHC class I molecules. Using this assay, lacZ activity of B3Z was comparable among B3Z cocultured with the OVA-transduced DCs, the OVA peptide (SIINFEKL)-pulsed DCs, and E.G7-OVA, an OVA transfectant of the T cell lymphoma EL4 (C57BL/6 background) (P b 0.01, Fig. 1D). This suggests that OVA was processed by the transduced DCs in a manner suitable for allowing presentation via their MHC class I molecules, and the ability with respect to an OVA epitope, SIINFEKL, was comparable to that of the peptide-pulsed DCs. In Vivo Comparison of OVA-Transduced DCs with OVA Peptide-Pulsed DCs We assessed the ability of the transduced DCs to induce CTLs against OVA-expressing tumors by inoculation of 5 105 DCs into C57BL/6 mice on Days 14 and 7 followed by implantation of 5 106 E.G7-OVA cells on Day 0 (Fig. 2A). Mice inoculated with the OVA peptide-pulsed DCs were unable to reject E.G7-OVA completely although they induced partial regression of the tumors compared with the tumors in mice without vaccine (P b 0.05). In contrast, mice inoculated with the OVA-transduced DCs completely rejected E.G7-OVA, while allowing progression of EL4 (Fig. 2B), suggesting that OVA-specific immunity was induced in the mice. To compare the anti-tumor effects of OVA-transduced DCs and OVA peptide-pulsed DCs, we inoculated 1 104 to 5 106 of these cells into mice twice (Days 14 and 7) prior to injection of 5 106 E.G7-OVA on Day 0 (Table 1). Approximately half of the mice that had received 1 105 transduced DCs showed rejection of the tumor, whereas all of the mice pretreated with 5 105 transduced DCs displayed complete rejection. On the other hand, none of the mice that had been inoculated

MOLECULAR THERAPY Vol. 13, No. 2, February 2006 Copyright C The American Society of Gene Therapy

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FIG. 1. Structure of the retroviral vector GCDN/OVA and characteristics of the OVA-transduced DCs. (A) The retroviral vector GCDNsapI/E contains the PCMV LTR with intact splice donor and splice acceptor sequences and the EGFP cDNA downstream of the internal ribosome entry site. The OVA cDNA was inserted between the BamHI and the XhoI sites to generate GCDN/OVA. Sequences present in the vector are labeled as follows: MoMLV, Moloney murine leukemia virus LTR; PCMV, PCC4 cell-passaged myeloproliferative sarcoma virus LTR; c+, packaging signal; S.D., splice donor; S.A., splice acceptor; IRES, internal ribosome entry site. (B) Morphological appearance of OVA-transduced DCs. The DCs were cytospun followed by May–Gruenwald–Giemsa staining. The scale bar represents 20 Am. (C) Surface markers and transduction efficiency of the transduced DCs. Transduction efficiency of DCs was determined by EGFP expression. Representative data are shown. (D) lacZ activation of B3Z by OVA-transduced DCs. Target cells (D, EL4; E, E.G7-OVA; 5, DCs; n, OVA peptide-pulsed DCs; x, OVA-transduced DCs) were incubated with 1 105 B3Z (effector cells) at various cell ratios as indicated. Absorption of each well at OD570 is indicated. *P b 0.01, compared with values for EL4 and DCs. n = 5 to 7.

with 1 or 4 106 peptide-pulsed DCs were able to reject the tumor, suggesting that the transduced DCs induced stronger anti-tumor effects against OVA-expressing tumors than the peptide-pulsed DCs when mice were inoculated with the same number of DCs. Only weak anti-tumor effects were observed in mice injected with the OVA peptides. Induction of OVA-Specific Cellular and Humoral Immunity by the Transduced DCs To characterize the marked anti-tumor effects elicited by the transduced DCs, we measured the number of CTLs against E.G7-OVA using the OVA tetramer technology (Fig. 3A). The prevalence of CD8+ T cells carrying a T cell receptor recognizing the OVA peptide (SIINFEKL) was not significantly different (P N 0.05) between mice inoculated with the peptide-pulsed DCs


and the transduced DCs. On the other hand, splenic CD8+ T cells of mice inoculated with the transduced DCs gave rise to three to four times more spots in the Lysispot assay than those obtained from mice inoculated with the peptide-pulsed DCs (Fig. 3B). Given that the number of spots in the Lysispot assay reflects the total number of CTLs against E.G7-OVA in inoculated mice, this result, together with the observation that the number of CTLs recognizing the OVA peptide (SIINFEKL) was approximately equal in both of these types of inoculated mice, suggested that CTL clones specific for different OVA epitope peptides were established in mice inoculated with the transduced DCs. Interestingly, mice inoculated with the transduced DCs produced anti-OVA antibodies of all IgG subclasses in amounts comparable to those produced after conventional immunization using FreundTs adjuvant (Fig. 4A), sug-

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FIG. 2. Active immunotherapy with OVA-transduced DCs against the OVA-expressing tumor E.G7-OVA. (A) Mice left untreated (o) or inoculated with 5 105 OVA peptidepulsed DCs (n) or OVA-transduced DCs (x) on Days 14 and 7 were implanted with 5 106 E.G7-OVA on Day 0. The tumor size was measured over time and represented as the tumor index. Results from four mice per group are presented. *P b 0.05 compared with the untreated group. (B) Mice left untreated (o) or inoculated with 5 105 OVA-transduced DCs (x) on Days 14 and 7 were implanted with 5 106 EL4 on Day 0.

gesting that OVA-specific CD4+ T cells were established in the mice. Furthermore, we also detected an OVAspecific IgG2a antibody suggesting the presence of OVA-specific helper T cell type 1 cells (Th1). This was also confirmed by intracellular staining demonstrating the presence of CD4+ T cells secreting IL-2 and interferon-g in response to in vitro restimulation by OVA-transduced DCs (Fig. 4B). We confirmed involvement of OVA-specific CD4+ T cells in the anti-tumor effects by tumor challenge experiments using MHC class II-deficient mice, in which anti-tumor effects were decreased compared with those in wild-type mice treated with OVA-transduced DCs, although the immunized MHC class II-deficient mice showed, to some extent, anti-tumor responses (P b 0.01, Fig. 4C). Taken together, these results showed that the transduced DCs

were capable of priming not only OVA-specific CD8+ but also CD4+ T cells in treated mice. Eradication of the Tumor by Immunization with the Transduced DCs Having demonstrated that inoculation of the transduced DCs elicited potent anti-tumor effects by induction of both cellular and humoral immunity, we next assessed the possibility of clinical applications of the transduced DCs by inoculating the OVA-transduced DCs into mice with preexisting E.G7-OVA (Fig. 5). Mice that had been implanted with 2 106 E.G7-OVA on Day 3 or 2 were given three successive peritumoral injections of 5 105 transduced DCs on Days 0, 2, and 4. This treatment resulted in eradication of the tumor mass and prevented progress of the tumors after rechallenge on Day 79

TABLE 1: The number of mice with complete rejection of E.G7-OVA Inoculated number of DCs: Transduced DCs Peptide-pulsed DCs Inoculated dose of peptide: Peptide

1 104 0 (1)a 0 (1) 12.5 Ag 0 (1)

5 104 0 (3) 0 (3) 25 Ag 0 (3)

1 105 5 (12) 0 (4) 50 Ag 0 (3)

5 105 4 (4) 0 (4) 100 Ag 1 (3)

Mice were inoculated with the indicated dose of OVA-transduced DCs, OVA peptide-pulsed DCs, or OVA peptide on Days Day 0. The evaluation of complete rejection (tumor index = 0) was performed on Day 14. ND, not determined. a The values in parentheses represent the total number of mice in each group.

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1 106 3 (3) 1 (3)

14 and

4 106 ND 1 (2)

5 106 ND 1 (1)

7, followed by implantation of 5 106 E.G7-OVA on


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FIG. 3. Induction of cellular immunity against E.G7-OVA. (A) The number of splenic CD8+ T cells recognizing the OVA peptide (SIINFEKL) in mice inoculated with OVA peptide-pulsed DCs (5) or OVA-transduced DCs (x) was measured by FACS with anti-CD8a antibody and OVA peptide tetramer. The y axis indicates the percentage of the peptide-specific CD8+ T cells in mice inoculated with the number of DCs indicated on the x axis. (B) The prevalence of total CTLs against EL4 or E.G7-OVA. Target cells (2 104 EL4/EGFP or E.G7OVA/EGFP) were incubated with 5 104 splenic CD8+ T cells from untreated mice (solid bars), mice treated with the peptide-pulsed DCs (dotted bars), or mice treated with the transduced DCs (hatched bars). Representative data from one of three independent experiments are shown.

(approximately 60 days after complete regression) after vaccination.

DISCUSSION DCs genetically engineered to both express TAAs and present antigenic epitope peptides through their MHC– peptide complex would be attractive agents for use in cancer immunotherapy [6,22]. In the present study, such DCs were generated by in vitro differentiation of mouse HSCs that had been retrovirally transduced with OVA cDNA. The DCs induced potent cellular and humoral immunity against an OVA-expressing tumor upon inoculation into syngeneic mice, with consequent eradication of the tumor. Stable expression and continuous presentation of antigenic peptides through the MHC–peptide complex of the transduced DCs and the consequent induction of multiple CTLs and tumor-specific helper T cells are


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theoretical advantages of the transduced DCs over peptide-pulsed DCs [27–31]. However, the results of previous studies relevant to this issue were inconclusive, probably because of the low transduction efficiency and the decline of transgene expression in immature cells due to gene silencing [29,32]. To address this issue, we used the gene-silencing-resistant retroviral vector GCDNsap to transfer the OVA cDNA into HSCs for preparation of the OVA-transduced DCs because Moloney murine leukemia virus (MoMLV)-based retroviral vectors are very susceptible to gene silencing of the transgene expression in HSCs. The GCDNsap vector is resistant to gene silencing for two reasons: first, it harbors the PCC4 cell-passaged myeloproliferative sarcoma virus (PCMV)-derived long terminal repeat (LTR), which is less methylated than the MoMLV-derived LTR, and second, it has mutations in the negative control region of the LTR and the negative responsible elements of the primer binding site so that the negative transcription factors YY1 and factor A cannot bind to the respective sites in the vector [35]. The vector packaged in the VSV-G envelope yielded high transduction efficiency of HSCs and maintenance of high OVA expression even after differentiation of the HSCs into mature DCs (Figs. 1B and 1C). Compared with previous reports in which the rates of transduction ranged from 0.1 to 20% [22], the transduction level of DCs obtained in this study was notably high. In addition, unlike adenovirus or lentivirus vectors [22,37,38], the retrovirus employed in the present study had no deleterious effects on differentiation of HSCs into mature DCs. Since the persistence of MHC class I/peptide complexes on the surface of DCs is critical in determining T cell responses, the high-level and stable expression of the full-length OVA cDNA resulted in more efficient induction of multiple CTLs that recognized different OVA epitope peptides in the tumor (Fig. 3B). It should be noted that attempts to document the presence of CTLs recognizing OVA peptides other than that used in this study were not made because of the unknown nature of those peptide sequences. Furthermore, the production of antibodies, including the IgG2a subclass (Fig. 4A), against OVA in mice inoculated with the transduced DCs suggested that OVA-specific Th1 had been established in vivo. This was also confirmed by the intracellular staining of cytoplasmic IL-2 and interferon-g in OVA-specific CD4+ T cells upon stimulation with OVA-transduced DCs (Fig. 4B). Although further experiments are needed to clarify how the OVA-specific Th1 is induced, one can imagine that OVA overexpressed by the transduced DCs was taken up by other DCs (Langerhans cells in dermis and/or DCs in lymph nodes etc.) or other antigen-presenting cells and consequently presented to CD4+ T cells through MHC class II molecules as an extracellular antigen. Recently, the importance of CD4+ T cells in

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FIG. 4. Involvement of OVA-specific CD4+ T cells in the anti-tumor effects. (A) IgG subclasses against OVA in mice with the indicated immunization were measured by enzyme-linked immunosorbent assay (OD450). Sera of mice immunized with OVA mixed with Freund’s adjuvant (OVA + FA) were used as a control. (B) OVA-specific Th1 was detected by Th1 intracellular cytokine staining. Splenic CD4+ T cells from mice immunized with or without OVA-transduced DCs were cocultured with the DCs, and intracellular Th1 cytokines were stained with anti-IL-2 or anti-interferon-g antibody. The patterns are shown for CD4+ gated cells. (C) Antitumor effects in MHC class II-deficient mice immunized with (w, n = 6) or without (o, n = 8) 1 105 OVAtransduced DCs (Days 14 and 7) were compared with those in wild-type mice immunized with (x, n = 8) or without ( , n = 8) the DCs. The tumor size of the mice was measured after implantation of 5 106 E.G7-OVA. *P b 0.05 compared with the nonimmunized MHC class II-deficient mice group. **P b 0.01 compared with the other groups. In the immunized wild-type mice group, three mice completely rejected E.G7-OVA.

.

anti-tumor immune responses has been reappraised and emphasized [39] because clinical trials using peptidepulsed DCs without loading of MHC class II-restricted peptides showed only weak anti-tumor responses. As might be expected, the importance of the OVA-specific CD4+ T cells in anti-tumor effects was shown in experiments using MHC class II-deficient mice, in which significant reduction of the anti-tumor effects was observed (Fig. 4C). Most importantly, the infusion of retrovirally transduced DCs both eradicated established tumors and

prevented the establishment of tumors that expressed the product of the vector transgene in a treatment study (Fig. 5). Clinically useful numbers of these antigenspecific DCs can be obtained by in vitro expansion of the transduced HSCs. While we are encouraged that this strategy may be used in the clinical setting, the utility of this DC vaccine should be evaluated for various cancers using appropriate TAAs such as HER2, WT1, and melanoma antigens. DCs genetically modified with the GCDNsap vector may represent a potent and promising new tool for cancer immunotherapy.

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FIG. 5. Eradication of the preexisting tumor by transduced DC vaccine. Mice implanted with 2 106 E.G7-OVA on Day 3 or 2 were left untreated ( , n = 2) or inoculated with 5 105 transduced DCs (x, n = 4). Transduced DCs were injected peritumorally on Days 0, 2, and 4. Mice inoculated with transduced DCs were again implanted with 2 106 E.G7-OVA on Day 79 (arrow).

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MATERIALS

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METHODS

Mice. C57BL/6 mice (7-12 weeks of age) were purchased from Nihon Clea (Tokyo, Japan) and Charles River Japan (Yokohama, Japan). MHC class IIdeficient mice (C57BL/6 background) were kindly provided by Dr. D. Mathis (Harvard Medical School) [40]. All experiments were performed under our institutional guidelines. Retroviral vector construction. The structure of the retroviral vector GCDNsap, which has the LTR and primer binding site derived from PCMV and dl587rev, respectively, and the method to produce recombinant retroviruses packaged in VSV-G were described elsewhere [35]. To construct the vector expressing OVA, a BamHI–XhoI fragment containing a full-length OVA cDNA was inserted into the corresponding sites of GCDNsap followed by insertion of an XhoI–ClaI fragment containing the internal ribosomal entry site/EGFP into the vector. The resultant vector was named pGCDNsap-OVA-I/E. The vector was converted to the corresponding retrovirus by transduction into 293gpg cells [41]. The virus titer was 9.0 106 infectious units/ml on Jurkat cells. Cell cultures. Packaging cell lines 293gp [42] and PG13 [43] were maintained in high glucose (4500 mg/L) Dulbecco’s modified EagleTs medium with 10% heat-inactivated fetal calf serum (FCS; Moregate BioTech, Bulimba, Australia), 2 mM l-glutamine, 100 U/ml penicillin G sodium, and 100 mg/ml streptomycin sulfate. 293gpg cells, which were genetically engineered to express VSV-G under the control of a tetracycline-inducible system, were maintained in Dulbecco’s modified Eagle’s medium with 10% FCS, 2 mM l-glutamine, 100 U/ml penicillin G sodium, 100 mg/ml streptomycin sulfate, 0.3 mg/ml G418, 2 Ag/ml puromycin, and 1 Ag/ml tetracycline. The lymphoma cell line EL4 (C57BL/6 background) [44], its OVA transfectant E.G7-OVA [45], and the T cell hybridoma B3Z [46], which was generated by fusion of an OVA MHC class I epitope (SIINFEKL)/H-2Kb-specific T cell clone with a lacZinducible cell line, were cultured in RPMI 1640 with 10% FCS, 2 mM lglutamine, 100 U/ml penicillin G sodium, 100 mg/ml streptomycin sulfate, and 50 AM 2-mercaptoethanol. 293gpg and B3Z cells were kindly provided by Drs. R. Mulligan (Harvard Medical School) and N. Shastri (University of California), respectively. All culture reagents except for FCS were purchased from Sigma–Aldrich (St. Louis, MO, USA). Generation of the OVA-transduced DCs. KL cells were isolated from mouse bone marrow as previously described [47]. Briefly, cells at low density (b1.077 g/ml) were stained with a biotinylated lineage-antibody mixture containing anti-mouse Gr-1 (RB6-8C5), Mac-1 (M1/70), B220 (RA3-6B2), CD4 (GK1.5), CD8a (53-6.7), and Ter-119 (Ter-119) monoclonal antibodies. Lineage+ cells were bound to streptavidin-magnetic beads (Dynabeads M-280 Streptavidin, Dynal Biotech, Oslo, Norway) and depleted with MPC-1 (Dynal). After depletion of lineage+ cells, cell preparations were incubated with mouse CD117-MicroBeads (Miltenyi Biotec, Gladbach, Germany) and c-KIT+ cells were isolated using miniMACS (Miltenyi). All antibodies except for mouse CD117-MicroBeads were purchased from BD PharMingen (San Diego, CA, USA). The purity of KL cells was more than 80%. KL cells (1–3 105) were cultured in StemPro34 (Invitrogen Life Technologies, Carlsbad, CA, USA) in the presence of 10 ng/ml mouse IL-3, 50 ng/ml mouse SCF, 100 ng/ml human thrombopoietin (provided by Kirin, Tokyo, Japan), 30 ng/ml mouse IL-6 (PharMingen), and 10 ng/ml human Flt3 ligand (R&D Systems, Minneapolis, MN, USA) in 24-well plates coated with human fibronectin fragment CH296 (RetroNectin; Takara Bio, Otsu, Japan) (Day 0). After 24 h of culture, cells were transduced with OVA by adding 50 Al of GCDN/OVA to the culture (Day 1). The next day, half of the culture medium was replaced with fresh culture medium containing the above cytokines and 50 Al of GCDN/OVA (Day 2) and the cultures were incubated for a further 3 h. The cells were then transferred into the wells of 24-well plates containing RPMI 1640 with 10% FCS in the presence of mouse IL-3, IL-6, and SCF, allowed to expand for an additional 5 days, and diluted to 5 to 10 105 cells/ml. The differentiation of the cells into DCs was induced following a method described by Inaba et al. [48] with some modifications. On Day 8,


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cells were washed and transferred to culture medium containing 10 ng/ml mouse IL-4 (eBioscience, San Diego, CA, USA) and 10 ng/ml mouse GMCSF (Kirin) at 7 to 10 105 cells/ml. To eliminate granulocytes floating in the culture medium, most of the culture medium was aspirated carefully so as not to remove DC precursors aggregating at the bottom on Days 10 and 12. Aggregates loosely adhering to the bottom were disassembled by pipetting and cultured at 5 105 cells/ml. On Day 14, floating cells were collected and used as mature DCs for subsequent experiments. Inoculation of the transduced DCs into mice. H-2Kb-restricted OVA peptide (SIINFEKL) was purchased from Hokkaido System Science (Sapporo, Japan). OVA peptide-pulsed DCs were prepared by incubation of DCs differentiated from KL cells with 10 AM OVA peptide for 2 h at 378C [9]. The OVA peptide vaccine (12.5 to 100 Ag) was prepared as 200 Al of mixture with incomplete Freund’s adjuvant (Sigma). In the vaccine studies, the vaccines above were subcutaneously injected in the left flank of mice on Days 14 and 7, and 5 106 E.G7-OVA or EL4 cells were subcutaneously injected in the right flank of the mice on Day 0. In the treatment study, mice that had been transplanted with 2 106 E.G7-OVA on Day 3 or 2 received 5 105 OVA-transduced DCs peritumorally by subcutaneous injection on Days 0, 2, and 4. The mice were rechallenged with 2 106 E.G7-OVA on Day 79. Tumor sizes were expressed as tumor index (square millimeters, length width) every 2 or 3 days for 2 weeks. Cell surface analyses. DCs were stained with phycoerythrin-conjugated anti-mouse CD11c (HL3; PharMingen); biotinylated anti-mouse CD40 (HM40-3), CD86 (GL1), and I-Ab (M5/114.15.2) (eBioscience); and streptavidin–allophycocyanin (PharMingen). After being washed with phosphate–buffered saline (PBS) containing 2% FCS (staining medium), stained cells were analyzed by flow cytometry (FACSCalibur; BectonDickinson, San Jose, CA, USA). For tetramer staining, the OVA epitope (SIINFEKL)-specific, phycoerythrin-conjugated T-Select H-2Kb OVA tetramer was purchased from MBL (Nagoya, Japan). Single-cell suspensions of splenocytes were resuspended with 100 Al of PBS and stained with fluorescein isothiocyanate-conjugated anti-mouse CD8a (53-6.7; PharMingen) and tetramer for 30 min on ice. After two washes with PBS, cells were analyzed with a FACSCalibur (Becton–Dickinson). Antigen presentation assay. The antigen presentation assay was performed following a reported protocol [49]. Briefly, EL4, E.G7-OVA, DCs, OVA peptide-pulsed DCs, and OVA-transduced DCs at 7.5 102 to 5 104 cells were cultured with 1 105 B3Z cells in 96-well U-bottom plates at 378C in 5% CO2. After incubation for 16 h, the cells were washed with PBS twice and then incubated with 100 Al of Z buffer (100 mM 2mercaptoethanol, 9 mM MgCl2, 0.125% Nonidet P-40, 0.15 mM chlorophenol red h-galactoside in PBS) at 378C for 4 h followed by the addition of 50 Al of stop buffer (300 mM glycine and 15 mM EDTA in doubledistilled water). Absorption of each well was read using a BioLumin-960 (Molecular Dynamics, Sunnyvale, CA, USA) (absorption wavelength 570 nm). All reagents were purchased from Sigma. Lysispot assay. The assay was performed following the original protocol described elsewhere [50] with modifications. EL4/EGFP and E.G7-OVA/ EGFP were prepared as target cells by transduction of the EGFP cDNA into EL4 and E.G7-OVA with the recombinant retroviruses expressing EGFP [34]. For preparation of effector cells, splenic CD8+ T cells from vaccinated mice were purified by magnetically positive selection. The EGFP-expressing target cells (2 104) were cocultured with CD8+ T cells in RPMI 1640 with 10% FCS, 2 mM l-glutamine, 100 U/ml penicillin G sodium, 100 mg/ ml streptomycin sulfate, 50 AM 2-mercaptoethanol, and 10 ng/ml mouse IL-2 (PharMingen) at 378C for 4 h in Multiscreen-IP ELISpot plates (Millipore, Bedford, MA, USA) coated with the antibody against GFP (Molecular Probes, Eugene, OR, USA). After three washes with PBS containing 0.05% Tween 20, 50 Al of biotinylated antibody against GFP (Abcam, Cambridge, UK) diluted at 1:1000 in PBS was added and incubated at room temperature for 1 h. After three washes, 50 Al of streptavidin–horseradish peroxidase (Sigma) diluted 1:1000 was added and incubated for an additional 1 h. Plates were carefully washed more than five times and spots in each well were developed using the AEC

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Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturerTs instructions. Enzyme-linked immunosorbent assay. Sera from mice vaccinated with 5 105 OVA peptide-pulsed DCs or OVA-transduced DCs were collected. As a positive control, mice were immunized with 20 Ag of recombinant OVA (Sigma) emulsified with complete FreundTs adjuvant (Sigma) on Day 0 and the same amount of OVA emulsified with incomplete FreundTs adjuvant on Day 7, and the sera were collected on Day 14. Nunc-Immuno plates (Nalge Nunc International, Rochester, NY, USA) were coated with 2 Ag/ml OVA at 48C for 16 h. After being washed three times, the plates were blocked with PBS containing 0.25% gelatin (Amersham Biosciences, Piscataway, NJ, USA) at room temperature for 1 h. After three washes, 50 Al of diluted serum was added and the incubation was continued for 2 h and the isotypes of OVA-specific antibodies were determined using a Mouse Immunoglobulin Isotyping ELISA Kit (PharMingen) according to the manufacturer’s instructions. Absorption of individual wells was read using a BioLumin-960 (Molecular Dynamics; absorption wavelength was 450 nm). Intracellular staining of Th1 cytokines. CD4+ T cells were isolated from splenocytes of mice vaccinated with or without OVA-transduced DCs twice by magnetically negative selection. One million CD4+ T cells were cocultured with 5 105 OVA-transduced DCs in 1 ml of RPMI 1640 with 10% FCS containing 10 ng/ml mouse IL-2 and BD GolgiStop (PharMingen) at 378C for 4 h. The cells were collected and resuspended with the staining medium and stained with biotinylated anti-CD4 (GK1.5; PharMingen) and streptavidin–allophycocyanin after Fcg receptors were blocked with antimouse CD16/32 (93; eBioscience) at 48C for 10 min. After being washed once, these cells were fixed and permeabilized with the BD Cytofix/ Cytoperm (PharMingen) at 48C for 20 min. After two washes with the BD Perm/Wash buffer (PharMingen), intracellular cytokines were stained with fluorescein isothiocyanate-conjugated anti-mouse IL-2 (JES6-5H4; eBioscience) or phycoerythrin-conjugated anti-interferon-g (XMG1.2; PharMingen). These stained cells were washed with the BD Perm/Wash buffer twice and staining medium once and then analyzed with a FACSCalibur (Becton–Dickinson). Statistical analyses. All statistical analyses in this study were performed with the Mann–Whitney U test. A probability of less than 0.05 (P b 0.05) was considered statistically significant.

ACKNOWLEDGMENTS The authors are thankful to Fabio Candotti, Richard A. Knazek (National Institutes of Health), and Shin-ichiro Honda (University of Tsukuba) for helpful and critical comments; to Diane Mathis, Richard C. Mulligan (Harvard Medical School), and Nilabh Shastri (University of California) for providing MHC class II-deficient mice, 293gpg, and B3Z cells, respectively; and to Ms. Naoko Okano for excellent secretarial assistance. This work was supported by a grant to M.O. from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. RECEIVED FOR PUBLICATION APRIL 26, 2005; REVISED SEPTEMBER 6, 2005; ACCEPTED SEPTEMBER 28, 2005.

REFERENCES 1. Steinman, R. M. (1991). The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9: 271 – 296. 2. Banchereau, J., and Steinman, R. M. (1998). Dendritic cells and the control of immunity. Nature 392: 245 – 252. 3. Banchereau, J., et al. (2000). Immunology of dendritic cells. Annu. Rev. Immunol. 18: 767 – 811. 4. Gallucci, S., Loloema, M., and Martinzer, M. (1999). Natural adjuvants: endogenous activators of dendritic cells. Nat. Med. 5: 1249 – 1255. 5. Liu, Y. (2001). Dendritic cell subsets and lineages, and their functions in innate and adaptive immunity. Cell 106: 259 – 262. 6. Gilboa, E., Nair, S. K., and Lyerly, H. K. (1998). Immunotherapy of cancer with dendritic-cell-based vaccines. Cancer Immunol. Immunother. 46: 82 – 87. 7. Fong, L., and Engleman, E. G. (2000). Dendritic cells in cancer immunotherapy. Annu. Rev. Immunol. 18: 245 – 273.

308

doi:10.1016/j.ymthe.2005.09.021

8. Ardavin, C., Amigorena, S., and Sousa, C. R. (2004). Dendritic cells: immunobiology and cancer immunotherapy. Immunity 20: 17 – 23. 9. Mayordomo, J. I., et al. (1995). Bone marrow-derived dendritic cells pulsed with synthetic tumor peptides elicit protective and therapeutic anti-tumor immunity. Nat. Med. 1: 1297 – 1302. 10. Nestle, F. O., et al. (1998). Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat. Med. 4: 328 – 332. 11. Banchereau, J., et al. (2001). Immune and clinical responses in patients with metastatic melanoma to CD34+ progenitor-derived dendritic cell vaccine. Cancer Res. 61: 6451 – 6458. 12. Ashley, D. M., Faiola, B., Nair, S., Hale, L. P., Bigner, B. D., and Gilboa, E. (1997). Bone marrow-generated dendritic cells pulsed with tumor extracts or tumor RNA induce anti-tumor immunity against central nervous system tumors. J. Exp. Med. 186: 1177 – 1182. 13. Wen, Y., Min, R., Tricot, G., Barlogie, B., and Yi, Q. (2002). Tumor lysate-specific cytotoxic T lymphocytes in multiple myeloma: promising effector cells for immunotherapy. Blood 99: 3280 – 3285. 14. Gong, J., Chen, D., Kashiwaba, M., and Kufe, D. (1997). Induction of anti-tumor activity by immunization with fusions of dendritic and carcinoma cells. Nat. Med. 3: 558 – 561. 15. Gong, J., et al. (2000). Activation of antitumor cytotoxic T lymphocytes by fusions of human dendritic cells and breast carcinoma cells. Proc. Natl. Acad. Sci. USA 97: 2715 – 2718. 16. Phan, V., et al. (2003). A new genetic method to generate and isolate small, short-lived but highly potent dendritic cell-tumor cell hybrid vaccines. Nat. Med. 9: 1215 – 1219. 17. Nair, S. K., et al. (2000). Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells. Nat. Med. 6: 1011 – 1017. 18. Heiser, A., et al. (2002). Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. J. Clin. Invest. 109: 409 – 417. 19. Alijagic, S., Moller, P., Artuc, M., Jurgovsky, K., Czarnetzki, B. M., and Schadendorf, D. (1995). Dendritic cells generated from peripheral blood transfected with human tyrosinase induce specific T cell activation. Eur. J. Immunol. 25: 3100 – 3107. 20. Arthur, J. F., et al. (1997). A comparison of gene transfer methods in human dendritic cells. Cancer Gene Ther. 4: 17 – 25. 21. Tu¨ ting, T., et al. (1998). Autologous human monocyte-derived dendritic cells genetically modified to express melanoma antigens elicit primary cytotoxic T cell responses in vitro: enhancement by cotransfection of genes encoding the Th1-biasing cytokines IL-12 and IFN-a. J. Immunol. 160: 1139 – 1147. 22. Jenne, L., Schuler, G., and Steinkasserer, A. (2001). Viral vectors for dendritic cell-based immunotherapy. Trends Immunol. 22: 102 – 107. 23. Bellone, M., Iezzi, G., Imro, M. A., and Protti, M. P. (1999). Cancer immunotherapy: synthetic and natural peptides in the balance. Immunol. Today 20: 457 – 462. 24. Rosenberg, S. A., Yang, I. C., and Restifo, M. P. (2004). Cancer immunotherapy: moving beyond current vaccines. Nat. Med. 10: 909 – 915. 25. Cranmer, L. D., Trevor, K. T., and Hersh, E. M. (2004). Clinical applications of dendritic cell vaccination in the treatment of cancer. Cancer Immunol. Immunother. 53: 275 – 306. 26. Schuler, G., Schuler-Thuener, B., and Steinman, R. M. (2003). The use of dendritic cells in cancer immunotherapy. Curr. Opin. Immunol. 15: 138 – 147. 27. Henderson, R. A., Nimgaonkar, M. T., Watkins, S. C., Robbins, P. D., Ball, E. D., and Finn, O. J. (1996). Human dendritic cells genetically engineered to express high level of the human epithelial tumor antigen mucin (MUC-1). Cancer Res. 56: 3763 – 3770. 28. Reeves, M. E., Royal, R. E., Lam, J. S., Rosenberg, S. A., and Hwu, P. (1996). Retroviral transduction of human dendritic cells with a tumor-associated antigen gene. Cancer Res. 56: 5672 – 5677. 29. Veerman, M. D., et al. (1999). Retrovirally transduced bone marrow-derived dendritic cells require CD4+ T cell help to elicit protective and therapeutic antitumor immunity. J. Immunol. 162: 144 – 151. 30. Lapointe, R., Royal, R. E., Reevesb, M. E., Altomare, I., Robbins, P. F., and Hwu, P. (2001). Retrovirally transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class II epitopes from the melanoma antigen glycoprotein 100. J. Immunol. 167: 4758 – 4764. 31. Buschenfelde, C. M., Metzger, J., Hermann, C., Nicklisch, N., Peschel, C., and Bernhard, H. (2001). The generation of both T killer and Th cell clones specific for the tumor-associated antigen HER2 using retrovirally transduced dendritic cells. J. Immunol. 167: 1712 – 1719. 32. Lindemann, C., et al. (2002). Down-regulation of retroviral transgene expression during differentiation of progenitor-derived dendritic cells. Exp. Hematol. 30: 150 – 157. 33. Kaneko, S., Onodera, M., Fujiki, Y., Nagasawa, T., and Nakauchi, H. (2001). Simplified retroviral vector GCsap with murine stem cell virus long terminal repeat allows high and continued expression of enhanced green fluorescent protein by human hematopoietic progenitors engrafted in non-obese diabetic/severe immunodeficient mice. Hum. Gene Ther. 12: 35 – 44. 34. Iwama, A., et al. (2002). Reciprocal roles for CCAAT/enhancer binding protein (C/


ARTICLE

doi:10.1016/j.ymthe.2005.09.021

35.

36. 37. 38. 39. 40. 41.

42.

43.

EBP) and PU.1 transcription factors in Langerhans cell commitment. J. Exp. Med. 195: 547 – 558. Suzuki, A., et al. (2002). Feasibility of ex vivo gene therapy for neurological disorders using the new retroviral vector GCDNsap packaged in the vesicular stomatitis virus G protein. J. Neurochem. 82: 953 – 960. Suzuki, A., et al. (2002). Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver. J. Cell Biol. 156: 173 – 184. Martin, F. C., III, Yu, Q., Wickham, T., Kovesdi, I., and Andreeff, M. (2000). Adenovirus as a gene therapy vector for hematopoietic cells. Cancer Gene Ther. 7: 816 – 825. Karlsson, S., Ooka, A., and Woods, N. B. (2002). Development of gene therapy for blood disorders by gene transfer into haematopoietic stem cells. Haemophilia 8: 255 – 260. Toes, R. E., Ossendrop, F., Offrunga, R., and Melief, C. J. (1999). CD4 T cells and their role in antitumor immune responses. J. Exp. Med. 189: 753 – 756. Cosgrove, D., et al. (1991). Mice lacking MHC class II molecules. Cell 66: 1051 – 1066. Ory, D. S., Neugeboren, B. A., and Mulligan, R. C. (1996). A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc. Natl. Acad. Sci. USA 93: 11400 – 11406. Burns, J. C., Friendmann, T., Driever, W., Burrascano, M., and Yee, J. (1993). Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc. Natl. Acad. Sci. USA 90: 8033 – 8037. Miller, A. D., Garcia, J. V., Suhr, N., Lynch, C. M., Wilson, C., and Eiden, M. V. (1991).


44. 45. 46.

47.

48.

49.

50.

Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J. Virol. 65: 2220 – 2224. Gorer, P. A. (1950). Studies in antibody response of mice to tumour inoculation. Br. J. Cancer 4: 372 – 379. Moore, M. W., Carbone, F. R., and Bevan, M. J. (1998). Introduction of soluble protein into the class I pathway of antigen processing and presentation. Cell 54: 777 – 785. Karttunen, J., Sanderson, S., and Shastri, N. (1992). Detection of rare antigenpresenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T cell antigens. Proc. Natl. Acad. Sci. USA 89: 6020 – 6024. Osawa, M., Hanada, K., Hamada, H., and Nakauchi, H. (1996). Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 273: 242 – 245. Inaba, K., et al. (1992). Generation of large numbers of dendritic cells from bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176: 1693 – 1702. Vabulas, R. M., Pircher, H., Lipford, G. B., Hacker, H., and Wagner, H. (2000). CpGDNA activates in vivo T cell epitope presenting dendritic cells to trigger protective antiviral cytotoxic T cell responses. J. Immunol. 164: 2372 – 2378. Snyder, J. E., Bowers, W. J., Livingstone, A. M., Lee, F. E., Federoff, H. J., and Mosmann, T. R. (2003). Measuring the frequency of mouse and human cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing. Nat. Med. 9: 231 – 235.

309