A novel tumor-vaccine cell therapy using bone marrow-derived dendritic cell type 1 and antigen-specific Th1 cells

Marimo Sato1, Kenji Chamoto1 and Takashi Nishimura1

1 Division of Immunoregulation, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-Ku, Sapporo 060-0815, Japan

Correspondence to: T. Nishimura; E-mail: tak24{at}imm.hokudai.ac.jp
Transmitting editor: M. Miyasaka


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Dendritic cell (DC)-based tumor-vaccine therapy is a rational strategy for tumor immunotherapy. However, using this protocol, it is still difficult to induce long-term regression in established tumor-bearing mice. To overcome this problem we developed a novel tumor-vaccine therapy, combining inactivated tumor cells with bone marrow-derived DC type 1 (BMDC1) and antigen-specific Th1 cells. BALB/c mice were intradermally inoculated with A20-OVA tumor cells expressing ovalbumin (OVA) as a model tumor antigen. After A20-OVA tumor mass became palpable (6–8 mm), mice were treated with DC-based vaccine therapy in various protocols. A complete cure of tumor-bearing mice was induced only when mice were repeatedly vaccinated with inactivated A20-OVA cells, OVA-pulsed BMDC1 and OVA-specific Th1 cells. Regression of tumor cells was associated with induction of Th1/Tc1-dominant antitumor immunity. Removal of one of these cellular components during vaccination resulted in failure to completely cure tumor-bearing mice. Moreover, BMDC2 cells could not replace the therapeutic effect of BMDC1 cells combined with Th1 cells. Thus, we propose a novel tumor-vaccine cell therapy using DC1 and Th1 cells.

Keywords: cell therapy, cytotoxic T lymphocyte, dendritic cell type 1, Th1, tumor immunotherapy


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system. These cells are considered natural adjuvants for inducing antitumor immunity in vivo (1). Indeed, immunization of mice with tumor vaccines plus DC or tumor antigen-pulsed DC is effective for inducing systemic protective antitumor immunity (2). Moreover, some promising therapeutic effects were also demonstrated in clinical studies with DC-based vaccines in human tumor patients (3). To establish a promising protocol of DC-based tumor-vaccine therapy in humans, it is critically important to establish an experimental protocol that completely cures established tumors in mice (4,5). However, effective protocols to completely cure mice bearing well-established tumors are not yet available.

In a previous paper (6) we demonstrated that Th1-dominant immunity exhibits strong protective antitumor immunity in vivo and is beneficial for inducing cytotoxic T lymphocyte (CTL) memory. Recent work has also demonstrated that initiation of Th1-mediated acquired immunity is linked with innate effector cells such as DC and NKT cells (1,7,8). In particular, DC subsets termed DC1 are beneficial for inducing Th1 cells from naive Th cells (9,10). In addition, we have demonstrated that Th1-cytokine-conditioned mouse myeloid DC derived from the bone marrow (BMDC1) are superior to Th2-cytokine-activated BMDC2 for inducing Th1 and allogenic CTL (11,12).

Based on these studies, we hypothesized that local administration of inactivated tumor cell vaccine combined with BMDC1 and Th1 cells may induce a tumor antigen-specific burst of Th1 cytokine production. In turn, a burst of Th1 cytokines may facilitate priming of tumor-specific CTL in vivo and migration of antitumor effector cells into tumor tissue. Here, we demonstrate that Th1 cells augment DC1-based tumor-vaccine therapy and completely cure established tumors in mice.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
BALB/c mice were obtained from Charles River Japan (Yokohama, Japan). BALB/c background RAG2–/– mice were kindly donated by Dr M. Ito (Central Institute for Experimental Animals, Kanagawa, Japan). Ovalbumin (OVA)323–339-specific I-Ad-restricted TCR transgenic mice (DO11.10) maintained on the BALB/c background were kindly donated by Dr K. M. Murphy (Washington University School of medicine, St Louis, MO) (6). All mice were female and used at 5–6 weeks of age.

Cytokines, mAb and antigens
IL-12 was kindly donated by Genetics Institute (Cambridge, MA). IL-2 was supplied by Ms Takuko Sawada (Shionogi Pharmaceutical Institute, Osaka, Japan). Anti-IL-12 mAb (C15.1 and C15.6) were a kind gift from Dr G. Trinchieri (Schering-Plough, Dardilly, France). Recombinant murine IL-4 and anti-asialo-GM1 antibodies were purchased from Wako Pure Chemical Industries (Osaka, Japan). Anti-IL-4 mAb (11B11) was purchased from ATCC (Rockville, MD). Phycoerythrin (PE)–anti-CD4 mAb, FITC–anti-CD45RB mAb, FITC–anti-CD8 mAb, purified anti-CD3 mAb, PerCP–anti CD3 mAb, FITC–anti-IFN-{gamma} mAb and anti-IFN-{gamma} mAb (R4-6A2) were purchased from PharMingen (San Diego, CA). Recombinant IL-3, IL-4, IFN-{gamma} and granulocyte macrophage colony stimulating factor (GM-CSF) were purchased from Peprotech (London, UK). OVA323–339 peptide was kindly supplied by Dr H. Tashiro (Fujiya, Hadano, Japan).

Generation of Th1 and Th2 cells
CD4+CD45RB+ naive T cells were isolated from nylon-passed spleen cells from a DO11.10 TCR transgenic mouse using a FACSVantage (Becton Dickinson, San Jose, CA) as reported previously (6). Purified CD4+CD45RB+ cells were stimulated with 10 µg/ml OVA323–339 peptide in the presence of mitomycin C (MMC)-treated BALB/c spleen cells, 20 U/ml IL-12, 1 ng/ml IFN-{gamma}, 50 µg/ml anti-IL-4 mAb and 20 U/ml IL-2 for Th1 development. Th2 cells were induced from the same naive Th cells in the presence of 1 ng/ml IL-4, 50 µg/ml anti-IFN-{gamma} mAb, 50 µg/ml anti-IL-12 mAb and 20 U/ml IL-2. At 48 h, cells were re-stimulated with OVA323–339 under the same conditions and used at 9–12 days of culture.

Preparation of BMDC1 and BMDC2
BM cells obtained from BALB/c mouse femora were cultured under three distinct culture conditions in 12-well plates (Costar, New York, NY) for 5 days (11,12). BMDC0 were induced by culture of BM cells (5 x 106 cells/well) in the presence of GM-CSF (20 ng/ml) and IL-3 (20 ng/ml). BMDC1 were induced by culture of BM cells in the presence of IFN-{gamma} (20 ng/ml) and IL-12 (20 U/ml) in addition to GM-CSF plus IL-3. BMDC2 were induced by culture of BM cells in the presence of IL-4 (20 ng/ml) in addition to GM-CSF plus IL-3. Three days after the initiation of culture, non-adherent lymphoid cells contaminating in BM cells were removed from the culture and replaced with fresh medium containing cytokine cocktails. DC harvested from 5- to 6-day-old cultures of BM cells were used as BMDC subsets (11,12). Converted BMDC1 were induced from BMDC0 by adding IFN-{gamma} into BMDC0 cultures at day 4 of culture. After 2 days of culture with IFN-{gamma}, cells were detached from the plates by vigorous pipetting after treatment with 0.25% trypsin in the presence of 0.5 mM EDTA solution.

Flow cytometry
The phenotypic characterization of DC and CTL was carried out using a FACSCalibur flow cytometer (Becton Dickinson) and CellQuest software. Detailed procedures for staining and sorting were described previously (6). Fluorescence data were collected with logarithmic amplification. For each sample, data from 10,000 volume-gated viable cells were collected. In some experiments, CD8+ T cells isolated from mixed lymphocyte cultures (MLC) by sorting with a FACSVantage (Becton Dickinson) were used for cytotoxicity assays. Mean fluorescence intensity was calculated using CellQuest software.

Intracellular cytokine expression
For the detection of cytoplasmic cytokine expression, cells stimulated with immobilized anti-CD3 mAb for 6 h in the presence of Brefeldin A were first stained with PerCP-conjugated anti-CD8 mAb, fixed with 4% paraformaldehyde, treated with permeabilizing solution (50 mM NaCl, 5 mM EDTA, 0.02% NaN3 and 0.5% Triton X-100, pH 7.5), and the fixed cells were then stained with PE-conjugated anti-IL-4 mAb and FITC-conjugated anti-IFN-{gamma} mAb for 45 min on ice. The percentage of cells expressing cytoplasmic IL-4 or IFN-{gamma} was determined by flow cytometry (FACSCalibur).

Serum IFN-{gamma} levels
Serum samples were obtained from mice and the level of IFN-{gamma} was measured using ELISA kits from Amersham (Little Chalfont, UK).

Cytotoxicity assay
The cytotoxicity mediated by CTL generated in MLC was measured by 4-h 51Cr-release assays as described previously (24). Tumor-specific cytotoxicity was determined using A20-OVA (H-2d) as target cells. As a control, DBA/2-derived P815 mastocytoma (H-2d) were used. The percent cytotoxicity was calculated as described previously (6).

Tumor-vaccine cell therapy
A20-OVA cells (2 x 106) were intradermally (i.d.) inoculated into BALB/c mice. When the tumor mass became palpable (6–8 mm), tumor-vaccine cell therapy was carried out under various conditions using MMC-treated A20-OVA (2 x 106) cells, OVA-pulsed BMDC1 (2 x 106), OVA-pulsed BMDC2 (2 x 106) and/or Th1 cells (5 x 106). Cells used for vaccination were mixed and i.d. injected into a distant site of the tumor mass. The antitumor activity mediated by the transferred cells was determined by measuring tumor size in perpendicular diameters. Tumor volume was calculated by the following formula: tumor volume = 0.4 x length (mm) x [width (mm)]2 (13). The mean of five mice per group is indicated in graphs.

Statistical analysis
Difference between the means of experimental groups were analyzed using Student’s t-test. Differences were considered significant where P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Preparation of large numbers of BMDC1 by conversion from BMDC0
It is now accepted that there are functionally distinct DC subsets (912). However, it remains unclear which DC subsets are beneficial for inducing antitumor activity against syngeneic tumors in vivo. To address this issue, we used BMDC subsets. As reported previously (11,12), BMDC1, which were induced from BM cells in the presence of Th1 cytokines + GM-CSF + IL-3, were superior for induction of Tc1 and Th1 cells, as compared with BMDC0 and BMDC2 subsets, which were induced with GM-CSF + IL-3 or GM-CSF + IL-3 + IL-4 respectively. Although BMDC1 have been considered promising tools for tumor immunotherapy, low yields have made it difficult to apply these cells to DC-based vaccine therapy in vivo. The yield of each BMDC subset harvested from 4-day cultures of BM cells (5 x 105 cells) is summarized in Table 1 (BMDC0, 3.1 ± 0.8 x 105 cells; BMDC1, 1.0 ± 0.3 x 105 cells; BMDC2, 2.3 ± 0.4 x 105 cells). Thus, BMDC1 showed the lowest yield among three distinct BMDC subsets. These results suggest that Th1 cytokines are beneficial for functional differentiation of DC, but inhibitory for the growth of DC. To induce expanded numbers of BMDC1, we attempted to induce these cells by conversion from BMDC0, which are easy to grow. BM cells were cultured with GM-CSF plus IL-3 (BMDC0 condition) for 4 days and then IFN-{gamma} was added into the culture to convert these cells into BMDC1. As shown in Table 1, the converted cells (BMDC0 -> BMDC1) exhibited a high yield (3.01 ± 1.0 x 105 cells). Moreover, the converted BMDC1 cells expressed higher levels of MHC class I, MHC class II and co-stimulatory molecules, and exhibited superior induction of IFN-{gamma}-producing CD8+ T cells (Tc1) and CD4+ T cells (Th1) in MLC compared with BMDC0 and BMDC2. We were unable to produce BMDC1 from BMDC2 incubated with IFN-{gamma} (data not shown). Thus, We are able to prepare increased (3-fold) numbers of BMDC1 with IFN-{gamma}. In the following set of experiments, we compared the adjuvant activity of BMDC1 and BMDC2 for inducing antitumor immunity in vivo.


View this table:
[in this window]
[in a new window]
 
Table 1. Functional characteristics of mouse BMDC subsets
 
Tumor-vaccine cell therapy
The DC-based tumor-vaccine therapy model was designed using BALB/c mice bearing A20-OVA tumor cells that express OVA as a model tumor antigen (6). We initiated tumor-vaccine therapy when the A20-OVA tumor mass became palpable (~8 mm). We utilized various combinations of MMC-treated A20-OVA cells, OVA-pulsed BMDC1 and OVA-specific Th1 cells. As shown in Fig. 1, treatment of tumor-bearing mice with i.d. injection of MMC-A20-OVA tumor vaccine (three injections at 2-day intervals) did not significantly inhibit tumor growth, compared with untreated mice. Treated mice died ~20 days after tumor inoculation. As reported previously (6), i.v. transfer of >2 x 107 OVA-specific Th1 cells induced complete tumor eradication, but i.d. injection of 5 x 106 Th1 cells was insufficient to induce strong antitumor activity. Although treatment of tumor-bearing mice with MMC-A20-OVA cells combined with OVA-pulsed BMDC1, OVA-pulsed BMDC2 or OVA-specific Th1 cells caused a slight inhibition of tumor growth, none of these combined therapies induced complete regression. However, when tumor-bearing mice were treated with MMC-A20-OVA cells combined with OVA-pulsed BMDC1 plus OVA-specific Th1 cells, tumor growth was greatly inhibited and eight out of 10 mice were completely cured from tumors (Fig. 1). Typical tumor-growth patterns in mice treated with various vaccine protocols are shown in Fig. 2. Augmentation of DC-based tumor-vaccine efficacy by Th1 cells was demonstrated when BMDC1, but not BMDC2, were included (Figs 1 and 2). In contrast to cells loaded with intact OVA proteins, OVA323–339 peptide-pulsed BMDC1 failed to cure mice, despite the fact that this protocol effectively inhibited tumor growth early after vaccination (data not shown). These results suggest that loading of BMDC1 with whole tumor protein antigen is superior for inducing potent antitumor activity. A single treatment of tumor-bearing mice with unpulsed BMDC1 combined with MMC-A20-OVA cells and Th1 cells was ineffective for inducing antitumor activity (Fig. 3), although repeated injection of Th1 cells and MMC-A20-OVA exhibited more efficient antitumor activity as described in Fig. 1. Thus, successful antitumor activity was observed only when three cellular components (OVA-pulsed BMDC1, A20-OVA and OVA-specific Th1) were included in the vaccine.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. A novel tumor-vaccine cell therapy using BMDC1 and Th1 cells. (A), BALB/c mice bearing a A20-OVA tumor mass (6–8 mm) were treated with or without (diamonds, solid lines) MMC-treated A20-OVA cells alone (diamonds, dashed lines), MMC-A20-OVA cells + OVA-pulsed BMDC1 (triangles), MMC-A20-OVA cells + Th1 cells (open circles, dashed lines), MMC-A20-OVA + Th1 + OVA-pulsed BMDC1 (solid circles) or MMC-A20-OVA + Th1 + OVA-pulsed BMDC2 (open circles, solid lines). Days of treatment indicated by arrows. Tumor volume was calculated as described in Methods. (B) Survival curves of A20-OVA tumor-bearing mice treated with various protocols described in (A).

 


View larger version (86K):
[in this window]
[in a new window]
 
Fig. 2. Rejection of tumor mass by DC1/Th1 tumor-vaccine therapy. Typical tumor growth patterns of A20-OVA tumor-bearing mice treated with various protocols. (A) None, (B) MMC-treated A20-OVA cells alone, (C) MMC-A20-OVA cells + Th1 cells, (D) MMC-A20-OVA cells + OVA-pulsed BMDC1, (E) MMC-A20-OVA cells + OVA-pulsed BMDC2, (F) MMC-A20-OVA cells + Th1 cells + OVA-pulsed BMDC1 and (G) MMC-A20-OVA cells + Th1 cells + OVA-pulsed BMDC2.

 


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3. Pulsing of BMDC1 with tumor protein antigen is essential for inducing a strong antitumor response. A20-OVA tumor-bearing mice were treated with none (solid diamonds), MMC-A20-OVA cells + Th1 cells + OVA-pulsed BMDC1 (solid circles), MMC-A20-OVA cells + Th1 cells + OVA-unpulsed BMDC1 (open circles), Th1 cells + OVA-pulsed BMDC1 (solid triangles) or Th1 cells + OVA-unpulsed BMDC1 (open triangles). Day of treatment indicated by arrow. *P < 0.01.

 
Mice cured from tumors by repeated DC1/Th1 tumor-vaccine therapy have increased tumor-specific Tc1 cells
To completely cure tumor-bearing mice, repeated DC1/Th1 tumor vaccination was essential because a single DC1/Th1 tumor vaccination was not able to completely cure tumor-bearing mice, whereas three treatments completely cured four out of five mice (Fig. 4A). In mice cured from tumor by repeated DC1/Th1 tumor vaccination therapy, we observed a marked elevation of serum IFN-{gamma} 24 h after the third vaccination (Fig. 4B). Consistent with these findings, we detected IFN-{gamma}-producing CD4+ T cells (Th1) and CD8+ T cells (Tc1) cells in these animals, but not in untreated control mice (Fig. 4C). Most (>95%) IFN-{gamma}-producing CD4+ T cells expressed KJ1-26, indicating those cells were derived from transferred Th1 cells. In mice that received a single therapeutic dose, little IFN-{gamma} production was observed (data not shown). Moreover, we demonstrated that antigen-specific CD8+ CTL were induced by in vitro re-stimulation of spleen cells obtained from the mice that received three injections of tumor vaccine (Fig. 4D). Such serum IFN-{gamma} elevation, augmented induction of IFN-{gamma}-producing CD8+ T cells and CTL generation was not observed in tumor-bearing mice treated with MMC-A20-OVA + BMDC1 (data not shown). We further showed that DC1/Th1 tumor-vaccine cell therapy does not completely cure of tumors in RAG2–/– T cell-deficient mice unless CD8+T cells were transferred into these animals (data not shown). These findings indicate that activation of CD8+ CTL in a tumor-bearing host is essential for curing mice of tumors by DC1/Th1 tumor vaccination therapy.



View larger version (41K):
[in this window]
[in a new window]
 
Fig. 4. Activation of type-1-dependent antitumor cellular immunity by repeated DC1/Th1 tumor-vaccine cell therapy in tumor-bearing mice. (A) A20-OVA tumor-bearing mice were treated with none (solid circles), DC1, Th1 and MMC-A20-OVA (DC1/Th1 tumor-vaccine cell therapy) once (open circles) or 3 times (open triangles). The number in parentheses indicates the percentage of cured mice among all tumor-bearing mice. The data represents mean ± SE of five mice. (B) Serum samples were obtained from tumor-bearing mice treated with DC1/Th1 tumor-vaccine cell therapy 24 h after final vaccine therapy. As a control, serum samples were obtained from untreated tumor-bearing mice. Serum IFN-{gamma} levels were measured by ELISA. The data represents mean ± SE of three mice. (C) Increase of IFN-{gamma}-producing Th1 and Tc1 cells by DC1/Th1 tumor-vaccine therapy. Spleen cells were prepared from control mice or mice treated with DC1/Th1 vaccine therapy three times. The percentages of IL-4- and IFN-{gamma}-producing cells were measured by intracellular staining. (D) Spleen cells obtained from untreated (triangles) or DC1/Th1-treated tumor-bearing mice (circles) were re-stimulated with OVA for 4 days. After culture, the cytotoxicity of CD8+ T cells against A20-OVA (solid symbols) or P815 tumor cells (open symbols) was measured by 4-h 51Cr-release assay. The data represents mean ± SE of three mice.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this paper, we demonstrate that tumor antigen-specific Th1 cells augment the therapeutic effect of DC-based tumor vaccination therapy (Figs 1–3). We also show that Th1 cytokine-activated BMDC1 are superior to Th2 cytokine- activated BMDC2 for completely curing tumor-bearing mice.

Since the gene(s) encoding tumor-rejection antigens were isolated, many investigators have tried to develop effective strategies for tumor-specific immunotherapy (14). For this purpose, tumor-antigen peptides were applied to tumor-vaccine therapy (15). However, clinical studies indicated that peptides can elicit antigen-specific CTL in vivo, but the increased frequency of CTL did not lead to tumor rejection in vivo (16). To increase the efficacy of tumor immunotherapy, DC have been combined with tumor peptides to induce tumor-specific immunity (1,3). Compared with peptide-vaccine therapy, DC-based vaccine therapy showed promising therapeutic activities in both mouse and human systems (35,17). However, it is still difficult to induce complete rejection of tumor mass by DC-based tumor-vaccine therapy alone. Thus, further improvement of DC-based tumor-vaccine therapy will be necessary for enhancing its efficacy. Although some reports have claimed that tumor antigen-pulsed DC can completely cure tumors in mouse models, DC-based vaccine therapy was carried out either before tumor inoculation or with a very small tumor burden (35,17). We have been unable to completely cure established tumors in mice by repeated injection of DC combined with inactivated tumor cells. These discrepancies might be due to several factors influencing the efficacy of DC-based vaccination: (i) antigen loading of DC, (ii) route of DC injection and (iii) origin of DC (1820).

It has been reported that tumor-associated antigen (TAA)-loaded DC may be superior for in vivo rejection of tumors expressing certain TAA than other strategies (e.g. naked DNA or peptide plus adjuvant) (21). Moreover, irradiated tumors are the most suitable for DC-based vaccination, as compared with tumor cells lysed by boiling or freeze–thaw (18). We vaccinated tumor-bearing mice by i.d. injection of BMDC1 combined with MMC-treated inactivated tumor cells, because the i.d. vaccination route with DC and tumor vaccine is superior to the i.v. tumor vaccination route (19). However, combined vaccination therapy with BMDC1 and MMC-treated tumor cells was ineffective (Figs 1 and 2).

In terms of DC origin, Th1 cytokine-activated BMDC1 are used to overcome negative factors in the tumor-bearing host. DC are crucial for inducing Th and CTL responses (1,2). In their immature state, DC express low levels of MHC and co-stimulatory molecules, but are efficiently equipped to sample antigen via receptor-mediated endocytosis or fluid-phase pinocytosis. In contrast, mature DC exhibit decreased antigen processing, but express high levels of MHC and co-stimulatory molecules (22). Recent studies have shown that activation and maturation of DC by cognate interaction with Th cells or certain stimuli (lipopolysaccharide, CpG and anti-CD40mAb) are essential for cross-presentation, which facilitates the priming of tumor-specific CTL (23). In particular, it has been shown that cognate interaction between DC and Th cells via CD40–CD40 ligand interaction is critically important for initiating cell-mediated immunity. During their interaction with Th cells, DC acquire a ‘license to kill’ which limits the duration of antigen-presenting activity of DC in vivo (24). However, it has been recently demonstrated that DC that interacted with Th1 cells express serine protease inhibitor 6 (SPI-6), which protects DC from CTL-induced apoptosis (25). However, DC that interacted with Th2 cells were sensitive to apoptosis because of low expression of SPI-6. These recent findings of DC biology greatly support the rationale to use Th1-cytokine-activated BMDC1 and Th1 cells for the potentiation of tumor-vaccine therapy.

As shown here (Table 1), BMDC1 induced from BMDC0 expressed high levels of MHC and co-stimulatory molecule (CD40 ligand, LFA-1 and B7-1) as well as mRNA for IFN-{alpha}, IFN-ß, IFN-{gamma}, IP-10 and IL-12 (data not shown), and are strongly phagocytic (dextran–FITC uptake) and strongly activate allogeneic CTL generation as well as BMDC1 (11,12). In contrast, Th2-cytokine-activated BMDC2 expressed low levels of MHC and co-stimulatory molecules, mRNA for IFN-ß, IFN-{gamma} and IP-10, and were poor activators of CTL. Surprisingly, treatment of BMDC2 with IFN-{gamma} resulted in increased expression of MHC molecules, but these cells were unable to support CTL generation (data not shown). Therefore, BMDC1 and BMDC2 cannot be simply classified into mature and immature DC, rather they should be defined as differentially conditioned ‘activated mature’ DC which have the distinct capacity to induce cell-mediated immunity. Consistent with their role in eliciting allogeneic CTL, we demonstrated here that BMDC1, but not BMDC2, when combined with Th1 cells, are suitable adjuvants for inducing tumor-specific immunity in vivo. This might be because BMDC1, which express high levels of MHC and co-stimulatory molecules, exhibit more efficient cross-presentation of OVA antigen or apoptotic MMC-A20-OVA tumor cells. If this is the case, BMDC1 efficiently present antigen to co-injected Th1 cells and subsequently to CTL-precursor cells. Based on previous reports (25,26), BMDC1 that interact with co-injected Th1 cells in vivo at the injection site may be allowed to escape apoptotic cell death after interacting with CTL. This permits DC to survive for a long period of time and may facilitate continuous stimulation of tumor-specific CTL in vivo. In fact, we demonstrated that IFN-{gamma}-producing CD8+ T cells and CTL are activated by repeated DC-based vaccination combined with Th1 cells and inactivated tumor cells (Fig. 4).

It is well known that tumor-bearing hosts suffer from strong immunosuppression. Many factors (e.g. regulatory T cells, suppressor cells, down-modulation of MHC molecules, overproduction of immunosuppressive factors, down-modulation of IL-2 response and IL-2 production, and poor DC function at the tumor site) explain the decreased immune responses in tumor-bearing hosts (20,27,28). Therefore, it is essential to overcome this strong immunosuppression to initiate tumor-specific immune responses in tumor-bearing hosts (1,29). As shown in Fig. 4, repeated injection of Th1 cells with BMDC1 and MMC-A20-OVA caused a marked elevation of serum IFN-{gamma} levels. Such IFN-{gamma} elevation was not induced by vaccination with BMDC1 and MMC-A20-OVA (data not shown). Therefore, co-injected Th1 cells may induce a cytokine storm at the immunization site, which acts as an initiator for DC-based tumor vaccination to induce tumor-specific CTL responses. It has been shown that Th1-dominant immunity accelerates the migration of both Th1 and CTL by up-regulating chemokine receptors (CCR5 and CXCR3) and integrins on T cells (30). Therefore, DC1/Th1 tumor-vaccine cell therapy appears to be a novel strategy to initiate Th1-dominant antitumor immunity, which may accelerate the infiltration of antitumor effector cells into the established tumor mass. We are currently investigating the detailed mechanisms underlying DC1/Th1 tumor-vaccine cell therapy.


    Acknowledgements
 
We would like to thank Dr Luc Van Kaer (Vanderbilt University School of Medicine, Nashville, TN) for reviewing this paper. We thank Dr M. Kobayashi (Genetics Institute) for her kind gift of IL-12 and Ms Takuko Sawada (Shionogi Pharmaceutical, Osaka, Japan) for providing IL-2. We also thank Ms Shinobu Miyamoto for secretarial assistance.


    Abbreviations
 
BM—bone marrow

BMDC—bone marrow-derived dendritic cell

CTL—cytotoxic T lymphocyte

DC—dendritic cell

GM-CSF—granulocyte macrophage colony stimulating factor

i.d.—intradermally

MMC—mitomycin C

MLC—mixed lymphocyte culture

OVA—ovalbumin

PE—phycoerythrin

SPI-6—serine protease inhibitor 6

TAA—tumor-associated antigen


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Steinman, R. M. and Dhodapkar, M. 2001. Active immunization against cancer with dendritic cells: the near future. Int. J. Cancer 94:459.[CrossRef][ISI][Medline]
  2. Ashley, D. M., Faiola, B., Nair, S., Hale, L. P., Bigner, D. D. and Gilboa, E. 1997. Bone marrow-generated dendritic cells pulsed with tumor extracts or tumor RNA induce antitumor immunity against central nervous system tumors. J. Exp. Med. 186:1177.[Abstract/Free Full Text]
  3. Thurner, B., Haendle, I., Röder, C., Dieckmann, D., Keikavoussi, P., Jonuleit, H., Bender, A., Maczek, C., Schreiner, D., Von den Driesch, P., Bröcker, E. B., Steinman, R. M., Enk, A., Kämpgen, E. and Schuler, G. 1999. Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J. Exp. Med. 190:1669.[Abstract/Free Full Text]
  4. Shimizu, K., Fields, R. C., Giedlin, M. and Mule, J. J. 1999. Systemic administration of interleukin 2 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines. Proc. Natl Acad. Sci. USA 96:2268.[Abstract/Free Full Text]
  5. Nikitina, Y. E. and Gabrilovich, D. I. 2001. Combination of {gamma}-irradiation and dendritic cell administration induces a potent antitumor response in tumor-bearing mice: approach to treatment of advanced stage cancer. Int. J. Cancer 94:825.[CrossRef][ISI][Medline]
  6. Nishimura, T., Iwakabe, K., Sekimoto, M., Ohmi, Y., Yahata, T., Nakui, M., Sato, T., Habu, S., Tashiro, H. and Ohta, A. 1999. Distinct role of antigen-specific T helper type 1 (Th1) and Th2 cells in tumor eradication in vivo. J. Exp. Med. 190:617.[Abstract/Free Full Text]
  7. Nishimura, T., Kitamura, H., Iwakabe, K., Yahata, T., Ohta, A., Sato, M., Takeda, K., Okumura, K., Van, L. K., Kawano, T., Taniguchi, M. and Nakui, M. 2000. The interface between innate and acquired immunity: glycolipid antigen presentation by CD1d-expressing dendritic cells to NKT cells induces the differentiation of antigen-specific cytotoxic T lymphocytes. Int. Immunol. 12:987.[Abstract/Free Full Text]
  8. Kadowaki, N. and Liu, YJ. 2002. Natural type I interferon-producing cells as a link between innate and adaptive immunity. Hum. Immunol. 63:1126.[CrossRef][ISI][Medline]
  9. Rissoan, M. C., Soumelis, V., Kadowaki, N., Grouard, G., Briere, F., De Waal Malefyt, R. and Liu, Y. J. 1999. Reciprocal control of T helper cell and dendritic cell differentiation. Science 283:1183.[Abstract/Free Full Text]
  10. Ohteki, T., Fukao, T., Suzue, K., Maki, C., Ito, M., Nakamura, M. and Koyasu, S. 1999. Interleukin 12-dependent interferon gamma production by CD8alpha+ lymphoid dendritic cells. J. Exp. Med. 189:1981.[Abstract/Free Full Text]
  11. Sato, M., Chamoto, K., Tsuji, T., Iwakura, Y., Togashi, Y. and Nishimura, T. 2001. Th1 cytokine-conditioned bone marrow-derived dendritic cells can bypass the requirement for Th functions during the generation of CD8+ CTL. J. Immunol. 167:3687.[Abstract/Free Full Text]
  12. Sato, M., Iwakabe, K., Kimura, S. and Nishimura, T. 1999. Functional skewing of bone marrow-derived dendritic cells by Th1- or Th2-inducing cytokines. Immunol. Lett. 67:63.[CrossRef][ISI][Medline]
  13. Ohta, S., Tsukamoto, H., Watanabe, K., Makino, K., Kuge, S., Kanai, N., Habu, S. and Nishimura, T. 1995. Tumor-associated glycoantigen, sialy Lewis(a) as a target for bispecific antibody-directed adoptive tumor immunotherapy. Immunol. Lett. 44:35.[CrossRef][ISI][Medline]
  14. Van der Bruggen, P., Traversari, C., Chomoes, P., Lurquin, C., De Plean, E., Van den Eynde, B., Knuth, A. and Boon, T. 1991. A gene encoding an antigen recognized by cytotoxic T lymphocytes on a human melanoma. Science 254:1643.[ISI][Medline]
  15. Wang, R. F. and Rosenberg, S. A. 1999. Human tumor antigens for cancer vaccine development. Immunol. Rev. 170: 85.
  16. Lee, K., Wang, E., Nielsen, M., Wunderlich, J., Migueles, S., Connors, M., Steinberg, S. M., Rosenberg, S. A. and Marincola, F. M. 1999. Increased vaccine-specific T cell frequency after peptide-based vaccination correlates with increased susceptibility to in vitro stimulation but does not lead to tumor regression. J. Immunol. 163:6292.[Abstract/Free Full Text]
  17. He, L., Feng, H., Raymond, A., Kreeger, M., Zeng, Y., Graner, M., Whitesell, L. and Katsanis E. 2001. Dendritic-cell-peptide immunization provides immunoprotection against bcrabl-positive leukemia in mice. Cancer Immunol. Immunother. 50:31.[ISI][Medline]
  18. Strome, S. E., Voss, S., Wilcox, R., Wakefield, T. L., Tamada, K., Flies, D., Chapoval, A., Lu, J., Kasperbauer, J. L., Padley, D., Vile, R., Gastineau, D., Wettstein, P. and Chen, L. 2002. Strategies for antigen loading of dendritic cells to enhance the antitumor immune response. Cancer Res. 62:1884.[Abstract/Free Full Text]
  19. Eggert, A. A., Schreurs, M. W., Boerman, O. C., Oyen, W. J., De Boer, A. J., Punt, C. J., Figdor, C. G. and Adema, G. J. 1999. Biodistribution and vaccine efficiency of murine dendritic cells are dependent on the route of administration. Cancer Res. 59:3340.[Abstract/Free Full Text]
  20. Gabrilovich, D. I., Corak, J., Ciernik, I. F., Kavanaugh, D. and Carbone, D. P. 1997. Decreased antigen presentation by dendritic cells in patients with breast cancer. Clin. Cancer Res. 3:483.[Abstract]
  21. Soares, M. M., Mehta, V. and Finn, O. J. 2001. Three different vaccines based on the 140-amino acid MUC1 peptide with seven tandemly repeated tumor-specific epitopes elicit distinct immune effector mechanisms in wild-type versus MUC1-transgenic mice with different potential for tumor rejection. J. Immunol. 166:6555.[Abstract/Free Full Text]
  22. Steinman, R. M. 2001. Dendritic cells and the control of immunity: enhancing the efficiency of antigen presentation. Mt Sinai J. Med. 68:106.[ISI]
  23. Schuurhuis, D. H., Laban, S., Toes, R. E. M., Ricciardi-Castagnoli, P., Kleijmeer, M. J., Van der Voort, E. I. H., Rea, D., Offringa, R., Geuze, H. J., Melief, C. J. M. and Ossendrop, F. 2000. Immature dendritic cells acquire CD8+ cytotoxic T lymphocyte priming capacity upon activation by T helper cell-independent or -dependent stimuli. J. Exp. Med. 192:145.[Abstract/Free Full Text]
  24. Lanzavecchia, A. 1998. Immunology. Licence to kill. Nature 393:413.[CrossRef][ISI][Medline]
  25. Medema, J. P., Schuurhuis, D. H., Rea, D., Van Tongeren, J., De Jong, J., Bres, S. A., Laban, S., Toes, R. E., Toebes, M., Schumacher, T. N., Bladergroen, B. A., Ossendorp, F., Kummer, J. A., Melief, C. J. M. and Offringa, R. 2001. Expression of the serpin serine protease inhibition 6 protects dendritic cells from cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1 and type 2 cells. J. Exp. Med. 194:657.[Abstract/Free Full Text]
  26. Shimizu, K., Thomas, E. K., Giedlin, M. and Mule, J. J. 2001. Enhancement of tumor lysate- and peptide-pulsed dendritic cell-based vaccines by the addition of foreign helper protein. Cancer Res. 61:2618.[Abstract/Free Full Text]
  27. Sakaguchi, S., Sakaguchi, N., Shimizu, J., Yamazaki, S., Sakihama, T., Itoh, M., Kuniyasu, Y., Nomura, T., Toda, M. and Takahashi, T. 2001. Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol. Rev. 182:18.[CrossRef][ISI][Medline]
  28. Whiteside, T. L. 1999. Signaling defects in T lymphocytes of patients with malignancy. Cancer Immunol. Immunother. 48:346.[CrossRef][ISI][Medline]
  29. Perales, M. A., Blachere, N. E., Engelhorn, M. E., Ferrone, C. R., Gold, J. S., Gregor, P. D., Noffz, G., Wolchok, J. D. and Houghton, A. N. 2002. Strategies to overcome immune ignorance and tolerance. Semin. Cancer Biol. 12:63.[CrossRef][ISI][Medline]
  30. Uesaka, Y., Yu, W., Mukai, T., Gao, P., Yamaguchi, N., Murai, M., Matsushima, K., Obika, S., Imanishi, T., Higashibata, Y., Nomura, S., Kitamura, Y., Fujiwara, H. and Hamaoka, T. 2002. A pivotal role for CC chemokine receptor 5 in T-cell migration to tumor sites induced by interleukin 12 treatment in tumor-bearing mice. Cancer Res. 62:3571.