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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Dendritic cell (DC) immunotherapy is a strong candidate for the treatment of incurable cancers especially malignant melanoma. Nevertheless, the proper guideline of DC immunotherapy does not exist. The absence of the guideline is also an obstacle to clinical trials of DC immunotherapy. So we conducted this study in order to develop an effective DC preparation method for immunotherapy in mouse malignant melanoma. Mouse bone marrow-derived DC were stimulated with tumour antigen alone or tumour antigen plus a cocktail (anti-CD40 antibody +TNF-α+ IL-1β) for 8, 24 or 48 h and the characteristics of these DC, such as surface molecules (CD40, CD80, CD86, MHC class II, CCR7), cytokines(IL-12, IFN-γ, and IL-10), DC-induced T cell proliferation in vitro, and the production of IFN-γ by those cells, were evaluated. Mice with melanoma were then treated with DC stimulated with tumour antigen alone and tumour antigen plus cocktail for 8 or 48 h. The tumour size and survival rate of these mice were then evaluated. (1) Beneficial clinical effects such as a reduction of tumour size and an increased survival rate were best observed in the group treated with DC stimulated for 8 h with tumour antigen plus cocktail. (2) The single prominent characteristic of DC stimulated for 8 h with tumour antigen plus cocktail was an elevated IL-12 secretion. The cytokine IL-12 was not secreted by other DC. Consequently, proper production of IL-12 was found to be an important requirement for DC used in immunotherapy of mouse melanoma.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Malignant melanoma is a potentially lethal cancer that arises from melanocytes present in the skin, mucosa, or epithelial surfaces of the eyes and ears, and its incidence has increased substantially over the past two decades [1, 2]. Although primary tumour excision can sometimes achieve complete remission, most melanomas are beyond surgical margins when diagnosed, and are usually resistant to chemotherapy and radiotherapy [3]. Consequently, many other therapeutic modalities are being investigated, one of them being dendritic cell (DC)-based immunotherapy [4, 5].

Dendritic cells (DC) are one of the most potent antigen-presenting cells [6]. DC residing in the skin or mucosa are in their immature state and have great phagocytic capacity. After antigen uptake, co-stimulator molecules are upregulated and the capacity to stimulate T cells is increased. Then stimulated T cells generate a potent host immunity to the antigen [7]. It has been reported that efficient antitumour immunity is generated by immature DC because immature DC phagocytose both necrotic and apoptotic tumour cells, and can mature in vivo [8]. The abilities of DC to phagocytose tumour cells in vivo and migrate to regional LN 24 h after injection have also been demonstrated. These findings suggest that DC can be used efficiently in cancer immunotherapy. Such DC-based immunotherapies have been attempted in mice and humans, and positive results have been obtained with many tumours, especially with malignant melanoma, B-cell lymphoma, colorectal cancer and prostate cancer [9–12].

Despite some favourable results, DC immunotherapy has yet to show acceptable, reasonable clinical effects. To enhance clinical effects, DC immunotherapy must have the ability to activate some effector cells such as cytotoxic T cells, helper T cells, B cells, natural killer (NK) cells, NK T cells or γδ T cells. In order to succeed in DC immunotherapy, sufficient considerations of both dendritic cellular factors and non-dendritic cellular factors are essential [13]. Currently, there is no consensus among the literature regarding the optimal guideline to follow in the development of DC immunotherapy. A successful clinical response with DC immunotherapy may not entirely depend on simply finding an optimal guideline of DC preparation. However, establishing an effective method of DC for immunotherapy must be the first step to successful DC immunotherapy.

In this study therefore, mouse bone marrow-derived DC were prepared and further stimulated with maturation factors for 8, 24 and 48 h. These several different types of DC were then injected into mice with malignant melanoma as a form of immunotherapy. By means of matching the functional characteristics of DC with clinical results obtained by DC immunotherapy, we attempted to define the effective DC preparation method for immunotherapy in mouse melanoma.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Mice.  C57BL/6 female mice (6–10 weeks old) were purchased from Daehan Biolink (Seoul, Korea) and housed in pathogen-free units at the Yonsei Medical Research Center.

Media and cytokines.  Complete medium (CM) consisting of RPMI 1640 (Invitrogen, Molecular Probes, Inc., Eugene, OR, USA) supplemented with 10% heat-inactivated FBS (Invitrogen), 100 IU/ml penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen), 0.1 mm nonessential amino acids (Sigma, St Louis, MO, USA), 1 mm sodium pyruvate (Sigma), 10 mm HEPES (Sigma) and 50 μm 2-mercaptoethanol (Sigma). DMEM (Invitrogen) and RPMI 1640 were used to culture hybridoma cell lines. Recombinant mouse GM-CSF (rmGM-CSF) or recombinant mouse IL-4 (rmIL-4) were purchased from Peprotech and used to generate DC. Maturation cocktail containing anti CD-40 antibody (BD Pharmingen, San Diego, CA, USA), TNF-α and IL-1β (Peprotech, Rocky Hill, NJ, USA) was used.

Preparations of bone marrow-derived DC.  Bone marrow cells were obtained from the tibias and femurs of C57BL/6 mice (6–8 weeks old) and depleted of erythrocytes using commercial lysis buffer (Sigma). Bone marrow cells were then treated with a mixture of antibodies (Abs) [anti-CD4 (GK1.5, TIB-207), anti-CD8 (53–6.72, TIB-105), anti-B220 (RA3-3A1/6.1, TIB-146), anti-I-Ab,d,q and I-Ed,k (M5/114.15.2, TIB-120), anti-erythrocytes, neutrophils and B cells (J11d.2, TIB-183); all were obtained from the ATCC] for 30 min at 4 °C, and then treated with rabbit complement (Cedarlane, ON, Canada) for 1 h at 37 °C. Cells were then layered onto lympholyte M (density: 1.0875 ± 0.0010 g/ml; Cedarlane) gradients, centrifuged, and the low-density interfaces were collected. Cells were washed three times with CM, and then incubated in CM supplemented with 50 U/ml GM-CSF and 5 U/ml IL-4 in 24 well plates at 7–10 × 105/well. On the second day of culture, floating cells were gently removed, and fresh medium containing GM-CSF and IL-4 was replaced. On day 4, floating cells were harvested and fresh medium containing GM-CSF and IL-4 was replaced and adherent cells were discarded. These harvested cells were then plated in new 24 well plates in CM supplemented with 50 U/ml GM-CSF and 5 U/ml IL-4 at 5 × 105/ml. On day 6, non-adherent and loosely adherent proliferating DC were collected and counted.

Flow cytometric analysis.  To identify surface molecules expressed on DC, cells were harvested on days 5 (D5), 6 (D6) and 7 (D7). Cells (6–10 × 105) were washed twice with 0.4% BSA/PBS and stained for 30 min at 4 °C with monoclonal Abs against CD11c, CD80, CD86 and MHC class II (I-Ab,d,q & I-Ed,k). Hamster IgG (Pharmingen) and rat IgG2a (Pharmingen) were used as isotype controls. All Abs were purchased from BD Pharmingen. After two washes with 0.4% BSA/PBS, secondary staining was performed using FITC-conjugated F(ab′)2 goat anti-rat Ig (Biosource, Camarillo, CA, USA) and FITC-conjugated anti-hamster IgG (BD Pharmingen). After 30 min, cells were washed twice with 0.4% BSA/PBS and resuspended in 400 μl of 0.4% BSA/PBS. Propidium iodide (Sigma) was added to exclude dead cells from the analysis. Flow cytometric analysis was performed on a FACSCalibur (Becton Dickinson, Mountain View, CA, USA).

Experimental design. In vitro: Prepared DC were grouped by an additional stimulation method and time. Groups 1, 2 and 3 were incubated for 8, 24 and 48 h, respectively, with no additional stimulation. Groups 4, 5 and 6 were incubated for 8, 24 and 48 h, respectively, under the stimulation of tumour antigen. B16F10 melanoma lysates (freeze-thawed three times, centrifuged at 700 g for 10 min, supernatant collected) were used as tumour antigen. DC were incubated with tumour antigen at 100 μg/ml. Finally, groups 7, 8 and 9 were incubated for 8, 24 and 48 h, respectively, with stimulation of tumour antigen + cocktail (anti CD-40 antibody 1 μg/ml, TNF-α 200 U/ml and IL-1β 5,000 U/ml).

In vivo: A malignant melanoma model was made by inoculating 2.5 × 105 B16F10 cells in 100 μl PBS subcutaneously into C57BL/6 mice (6–10 weeks old). Six days later, 50 mice were divided into five groups: (i) injected with PBS control (10 mice, group I), (ii) injected with DC stimulated for 8 h with tumour antigen (10 mice, group II), (iii) injected with DC stimulated for 48 h with tumour antigen (10 mice, group III), (iv) injected with DC stimulated for 8 h with tumour antigen plus cocktail (10 mice, group IV), (v) injected with DC stimulated for 48 h with tumour antigen plus cocktail (10 mice, group V). All stimulated DC (1 × 105cell) were injected directly into the melanoma. After the first intratumoral injection, another two intratumoral injections of DC were performed at 2-day intervals.

Measurement of tumour size and survival rate.  After DC immunotherapy with each group of mice, tumour long and short axes were measured with callipers (Mitutoyo, Kawasaki, Japan) at 2- to 3-day intervals. Tumour volumes were calculated using the formula V = 1/2 ×A2 × B, where A is the length of the short axis and B is that of the long axis. Surviving mice were counted until all group I mice had succumbed. Survival rates were recorded as percentage survivals.

Delayed-type hypersensitivity (DTH) assay.  Seven days after the third injection of DC, each mouse received 300 μg of tumour lysate (suspended in 50 μl of PBS) subcutaneously injected into the right footpad area. As a negative control, an identical amount of PBS was also injected into the left footpad of the same mouse. After 48 h, the extent of swelling was measured using callipers (Mitutoyo). Results were reported as the thickness difference (in millimetres) between both footpads.

T-cell proliferation assay.  Splenocytes were obtained from each experimental group and commercial erythrocyte lysis buffer was added. After 5 min, the reaction was stopped by adding CM, then washed twice with CM. Triplicate samples of 1.5 × 105 cells in CM were seeded in 96 well round-bottom plates (Corning, New York, NY, USA). Splenocytes (responder: R) were stimulated with B16F10 cells (stimulator: S) treated with mitomycin c and irradiation (30,000 rad) at a S:R ratio of 1:50. Splenocytes from untreated mice were used as a negative control. The cells were incubated at 37 °C for 96 h, then 1 μCi/well [3H] thymidine was added for another 16 h. Cells were harvested onto glass-fibre filters using a Harvester 96 (Tomtec, Hamden, CT, USA), and [3H] thymidine incorporation (CPM) was counted on a scintillation counter (Wallac, Turku, Finland).

ELISA.  To measure the IFN-γ secreted by splenocytes, splenocytes were stimulated with B16F10 cells and prepared as described above. Quantities of secreted IFN-γ were measured by ELISA using an OptEIA set (BD OptIEA mouse ELISA kit; BD Biosciences, San Jose, CA, USA) as described by the manufacturer. Briefly, anti-mouse IFN-γ Abs in 0.1 m pH 9.5 sodium carbonate (100 μl/well) were plated in 96 well plates (Corning) at 100 μl per well and incubated overnight at 4 °C. After washing, blocking solution (10% FBS/PBS) was added at 200 μl per well and incubated for 1 h at room temperature (RT). After washing, culture supernatants were plated at 100 μl per well and incubated for 2 h at RT. After washing, a mixture of biotinylated anti-mouse IFN-γ Ab and streptavidin-HRP reagent were plated at 100 μl per well, and then incubated for 1 h at RT. After washing, substrate solution (100 μg/ml tetramethyl benzidine and 0.003% H2O2) was added at 100 μl per well and incubated for 30 min, at RT, in the dark, followed by 4 n HCl. OD450 was measured on an ELISA Reader (Molecular Devices, Sunnyvale, CA, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Analysis of DC surface molecules

Erythrocyte-, lymphocyte- and neutrophil-depleted bone marrow cells from C57BL/6 mice were incubated with rmGM-CSF and rmIL-4 for 6 days. After additional stimulation, the surface expressions of CD11c, CD40, CD80, CD86, MHC class II and CCR7 on DC were examined in each DC group (Fig. 1). Upon flow cytometric analysis, moderate expression of CD11c (60–70%) was identified, which indicated that the DC had been generated from bone marrow cells stimulated with rmGM-CSF and rmIL-4 (data not shown). DC stimulated for 48 h with tumour antigen plus cocktail showed moderate expression of CD80, CD86 and MHC class II. However, DC cultured for 48 h with no simulation, or with tumour antigen alone, showed only weak expressions of CD80, CD86 and MHC class II.

image

Figure 1.  The surface phenotypes of each stimulated DC group were examined by flow cytometry using CD40, CD80, CD86, MHC class II and CCR7. Only CD11c-gated DC were used for flow cytometric analysis. The expression of surface molecules (CD80, CD86, MHC II) was increased in DC stimulated for 48 h with tumour antigen plus cocktail.

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Analysis of intracytoplasmic cytokines of DC

Intracytoplasmic cytokines (IL-12p40/p70, IL-10 and IFN-γ) of each DC group were examined by flow cytometric analysis. However, those cytokines were not stained in any experimental group (data not shown).

Analysis of cytokines secreted by DC

Cytokines secreted from DC were measured by ELISA (Fig. 2). IL-12p70 was detected in the tumour antigen plus cocktail stimulated group. DC stimulated for 8 h with tumour antigen plus cocktail showed marked secretion of IL-12p70 (2,065 pg/ml). As incubation time passed, the amount of secreted IL-12p70 decreased (24 h 1,120 pg/ml and 48 h 598 pg/ml). However, IL-12p70 was not detected in the control and the tumour antigen-only stimulated groups. No groups showed IFN-γ or IL-10 secretion (data not shown).

image

Figure 2.  The amount of secreted IFN-γ, IL-12p70 and IL-10 was measured by ELISA, which was performed as described in the OptEIA (Biosciences) catalogue. OD450 was measured on an ELISA Reader (Molecular Devices). The DC conditions for ELISA measurements were 7–10 × 105 cell/ml. DC stimulated for 8 h with tumour antigen plus cocktail showed more IL-12p70 secretion (2,065 ± 22 pg/ml) than DC stimulated for 24 h (1,120 ± 16 pg/ml) or 48 h (598 ± 22 pg/ml).

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T-cell proliferation assay

Isolated T cells from the spleen were used for T-cell proliferation analysis. The strongest T cell proliferation (7,253 cpm) was observed with DC stimulated for 8 h with tumour antigen plus cocktail. Elevated T cell proliferative reactions were also observed with DC stimulated for 24 or 48 h with the same methods (2,943 and 2,983 cpm respectively) (Fig. 3).

image

Figure 3.  Three separate experiments were performed. DC stimulated for 8 h with tumour antigen plus cocktail showed more T-cell proliferation (7,253 cpm) than DC stimulated for 24 h (2,943 cpm) or 48 h (2,983 cpm).

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Analysis of tumour size and mouse survival rate

Nineteen days after B16F10 injection to mice, tumour volume reduction was evident in the group treated with DC stimulated for 8 h with tumour antigen plus cocktail (group IV), which showed a much reduced tumour volume (695 ± 514 mm3) than the control (group I, 2,020 ± 808 mm3) (Fig. 4A). Mice treated with DC stimulated for 8 h with tumour antigen only (group II) also showed a tumour volume reduction.

image

Figure 4.  (A) Tumour volume was reduced in the group treated with DC stimulated for 8 h with tumour antigen plus cocktail (695 ± 514 mm3, control 2,020 ± 808 mm3). (B) The survival rate of mice was increased in the group treated with DC stimulated for 8 h with tumour antigen plus cocktail (66 days, control 32 days).

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Mice treated with DC stimulated for 8 h with tumour antigen plus cocktail showed the best survival rate (66 days, control 32 days) (Fig. 4B). The groups treated with DC stimulated with tumour antigen alone also showed increased survival rates (8 h at 46 days and 48 h at 42 days), but a survival benefit was not observed in mice treated with DC stimulated for 48 h with tumour antigen plus cocktail (33 days).

Delayed-type hypersensitivity

All study groups produced a DTH reaction when compared with the control group. The groups treated with 8 h-stimulated DC showed more potent DTH than 48 h-stimulated DC-treated groups. The most potent DTH reaction (1.0167 mm) was observed in the group treated with DC stimulated for 8 h with tumour antigen plus cocktail (control, 0.1033 mm) (Fig. 5).

image

Figure 5.  Seven days after the third DC injection, each mouse received 300 μg of tumour lysate subcutaneously injected into the right footpad area. As a negative control, an identical amount of PBS was also injected into the left footpad of the same mouse. After 48 h, the extent of swelling was measured using callipers. Results are reported as the differences in thickness (in millimetres) between both footpads.

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T-cell proliferation assay after DC immunotherapy

All study groups induced T cell proliferation in vitro when compared with control. Similar to the DTH reaction, 8 h-stimulated DC-treated groups showed stronger T-cell proliferation in vitro than the 48 h-stimulated DC-treated groups. The strongest T-cell proliferation (7,800 cpm) was observed in the group treated with DC stimulated for 8 h with tumour antigen plus cocktail (control, 2,572 cpm) (Fig. 6A).

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Figure 6.  (A) Eight hour-stimulated DC-treated groups show stronger T cell proliferation than 48 h-stimulated DC-treated groups. (B) IFN-γ secretion is more increased in 8 h-stimulated DC-treated groups than in 48 h-stimulated DC-treated groups.

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Cytokine secretion after DC immunotherapy

Similar to the DTH and T-cell proliferation assays, IFN-γ secretion was increased more in groups subjected to 8 h-stimulated DC than 48 h-stimulated DC. The highest IFN-γ secretion (30,148 pg/ml) was observed in the group treated with DC stimulated for 8 h with tumour antigen alone (Fig. 6B).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

In this study, mouse bone marrow-derived DC were generated and further stimulated with tumour antigen alone or tumour antigen plus cocktail (anti-CD40 antibody + TNF-α + IL-1β) for 8, 24 or 48 h respectively. The characteristics of DC, such as surface molecules (CD40, CD80, CD86, MHC class II, and CCR7), cytokines (IL-12, IFN-γ, and IL-10) produced by DC, DC-induced T cell proliferation in vitro and the production of IFN-γ by those T cells were then evaluated.

Furthermore, mice with malignant melanoma were treated with DC stimulated with tumour antigen alone or tumour antigen plus cocktail for 8 or 48 h respectively. Tumour size and survival rate were then evaluated. By means of matching the functional characteristics of DC with the clinical efficacy of DC immunotherapy, we attempted to determine the effective method of DC for DC immunotherapy in mouse melanoma.

The best clinical responses in terms of tumour volume reduction and increased survival rate were observed with DC stimulated for 8 h with tumour antigen plus cocktail. DC simulated for 8 h with tumour antigen alone also showed improved clinical responses, but these responses were weaker than those induced by DC stimulated with both tumour antigen and cocktail. Thus, we examined the characteristics of DC stimulated for 8 h with tumour antigen plus cocktail. These DC expressed more IL-12 than other groups. An in vitro T cell proliferation assay showed the strongest response with DC stimulated for 8 h with tumour antigen plus cocktail. IFN-γ secretion by DC-stimulated T cells was greater than other groups (data not shown). Immunological monitoring such as DTH, T-cell proliferation and IFN-γ secretion by T cells in vaccinated mice was also performed to compare the immune responses to clinical responses. All immunological monitoring methods showed that 8 h-stimulated DC-treated groups had stronger reactions than 48 h-stimulated DC-treated groups. With all of these functional characteristics, clinical responses and immunological reaction results, we concluded that proper secretion of IL-12 by DC is an effective method for DC immunotherapy.

In this study, the surface molecules (CD80, CD86 and MHC class II) were expressed the most in DC stimulated for 48 h with tumour antigen plus cocktail. However, clinical responses of this group were weaker than those induced by DC stimulated for 8 h with the same method. It is not fully known whether the status of DC maturation is a major indicator of clinical efficacy. In some clinical studies, T cell stimulatory capacities of immature DC are shown to be less, compared with mature DC [14]. However, other clinical trials indicate immature DC are superior to mature DC, at least with regard to the induction of T-cell responses [15, 16]. Nevertheless, consensus regarding the activation status of DC for effective DC immunotherapy has not been reached. Generally, DC stimulated with maturation factors are thought to be more potent APC than immature DC [17, 18]. However, the APC function of DC is thought to be limited to the later development of an immune response to avoid improper or autoimmune reaction. Several studies have shown that newly activated DC express cell surface molecules at high levels and produce some factors required for T cell activation. However, DC activated for long periods by maturation stimuli may not be functional immunologically [19–22].

In this study, IL-12 secretion was detected at a higher level in DC stimulated for 8 h with tumour antigen plus cocktail. However, control DC and tumour antigen alone stimulated DC showed no IL-12 secretion regardless of activation time. IL-12 is known to be a cytokine that is involved directly in the generation of CTL responses [23]. One study showed that after in vitro activation by LPS, as well as by poly(I)-poly(C) and TNF-α plus IL-1β, DC produce IL-12 only transiently, with a peak between 5 and 8 h, and complete extinction after 18 h, the time at which they become refractory to further stimulation by CD40L [22]. There are similar results in the literature using microarray techniques [24, 25]. It is more intriguing that DC activated for 8 h preferentially induced Th1 responses, but DC activated for 48 h induced Th2 responses and non-polarized T cells [22]. Eight hour-stimulated DC were previously observed to produce IL-12 maximally and move to regional lymphoid tissue efficiently [26]. It thus appears that 8 h-stimulated DC induce CD4+ T-cell activation [26], possibly Th1 polarization [27], and potent CTL [28, 29]. The so-called ‘gold standard’ mature DC (cultured in IL-4 and GM-CSF in combination with IL-1β, IL-6, TNF-α and PGE2) are known to have this handicap because of their low or absent production of IL-12 [30], and the concern that fully maturated DC are unable to induce immune responses properly [22]. These problems are supported by some studies using so-called mature ‘gold standard’ DC, which showed relatively low levels of clinical responses [31].

To induce an Ag-specific T-cell response, DC maturation is essential [32]. Many agents have been used to induce DC maturation experimentally such as monocyte-conditioned medium [33], cytokine mixture (consisting of TNF-α, IL-6, IL-1β and PGE2) [34], poly(I:C) (a synthetic analogue of dsRNA) [35], CpG oligonucleotides [36, 37] and LPS [38]. Poly(I:C), CpG oligonucleotides and LPS affect the target cells by binding to endogenous TLR (TLR3, 9 and 4) [35, 39–41]. LPS-induced DC maturation has been shown to markedly enhance the ability of Ag-loaded DC to stimulate an Ag-specific T cell response in vitro and in vivo animal models [32, 42, 43]. Nevertheless, due to toxicity (potentially leading to toxic shock) and batch-to-batch variability, the use of LPS for the maturation of DC in clinical immunotherapy is limited [44]. In this study, DC were also stimulated with tumour antigen plus LPS for 8, 24 or 48 h. The expression of surface marker on those DC was higher than tumour antigen alone or tumour antigen plus cocktail-stimulated-DC. IL-12 secretion was also increased to a greater level than DC stimulated for 8 h with tumour antigen plus cocktail. Especially, intracytoplasmic IL-12 was detected at a higher level in DC treated with tumour antigen plus LPS for 8 h, whereas IL-12 was nearly undetectable in DC treated with tumour antigen plus cocktail for 24 or 48 h. Ultimately, however, DC vaccines have been investigated with regard to human disease, especially human cancer. Though the use of LPS for DC maturation is expected to enhance Ag-specific immunity [33], other substitutable clinically safe stimulation factors need to be developed. Recently, in preparing a prophylactic melanoma vaccine, MART-1 (melanocyte/melanoma antigen recognized by T cells-1) expressing whole recombinant yeast was used for safety [45]. And, in order to lower IL-12 toxicity in clinical trials, IL-12-expressing transduced mesenchymal stem cells were used as a method for delivery [46].

In summary, mouse bone marrow-derived DC were generated using different stimulation methods and incubation times. DC immunotherapy on a malignant melanoma mouse model was then performed. After DC immunotherapy, functional characteristics of DC that showed the best clinical responses including reduction in tumour size and increased survival rates were evaluated. DC stimulated for 8 h with tumour antigen plus cocktail showed the best clinical responses. Moreover, those DC had elevated IL-12 secretion whereas others showed no IL-12 secretion. Thus, proper production of IL-12 is shown to be an important requirement for DC used in immunotherapy of mouse melanoma.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

This research was supported by a grant (04092-495) from the Korean Food and Drug Administration. We declare that our experiments comply with the current laws of the Republic of Korea inclusive of ethics approval.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References