ESAT-6-gpi DNA Vaccine Augmented the Specific Antitumour Efficacy Induced by the Tumour Vaccine B16F10-ESAT-6-gpi/IL-21 in a Mouse Model

Authors


Correspondence to: Professor J. Dou, Department of Pathogenic Biology and Immunology, Medical School, Southeast University, # 87 Ding Jiaqiao Rd. Nanjing 210009, China.

E-mail: njdoujun@yahoo.com.cn

Abstract

In this study, we hypothesized that the mice immunized with the glycosylphosphatidylinositol (GPI) anchored 6-kDa early-secreted antigenic target (ESAT-6) DNA vaccine (ESAT-6-gpi) and the tumour vaccine B16F10-ESAT-6-gpi/IL-21 might significantly enhance immune responses and antimelanoma efficacy. Our experimental results indicated that the anti-ESAT-6 antibody induced by the DNA vaccine ESAT-6-gpi bound ESAT-6 to the surface of tumour vaccine to activate a complement classical pathway and resulted in the B16F10 tumour cell lysis and apoptosis, which served as a potential trigger for breaking melanomatous immune tolerance to elicit an initiation of natural antimelanoma immunity. Our innovative approach of using the DNA vaccine ESAT-6-gpi priming and the tumour vaccine B16F10-ESAT-6-gpi/IL-21 boosting induced strong antimelanoma immunity that inhibited melanomatous growth. These findings highlighted the DNA vaccine ESAT-6-gpi as an immune enhancer to augment the immune efficacy of the tumour vaccine B16F10-ESAT -6-gpi/IL-21 against melanoma in a mouse model.

Introduction

Melanoma is a malignant skin tumour and the incidences of this tumour have been rising rapidly. When a tumour is metastatic, the prognosis is poor because metastatic melanoma is highly resistant to conventional therapies including surgery and chemotherapy. There are no known effective therapies for advanced melanoma at present [1, 2]. On the other hand, melanoma is a prototype of immunogenic tumour to which various types of immunotherapy have been applied extensively over the past decades [2]. Melanoma vaccines are designed for the purpose of immune modulation and its subsequent antimelanoma effects during an active specific immunotherapy [1, 3]. Unfortunately, the malignant melanoma is a low immunogenic tumour incapable of stimulating potent antitumour immune responses. Melanoma vaccines against melanoma require immune tolerance break because melanoma-associated antigens used in vaccine formula are mostly self-antigens [4-6]. To overcome the low immunogenic limitation of melanoma vaccine developed, a modified melanoma vaccine with heterogeneous antigen tolerance break and cytokine potent activator might enhance the immunogenicity of melanoma vaccine and induce melanoma-specific immune responses [7]. Evidence from past studies has indicated that the vaccination with DNA vaccine is a rapidly developing method for inducing immune responses that include effective cytotoxic T lymphocytes (CTL) and antibody responses[8, 9]; however, the immune response is not as strong as that from the traditional protein vaccines. Nonetheless, the efficacy of DNA vaccines may be enhanced by modifying a gene that codes tumour suppressor molecule or cytokine or heterogeneous antigen, etc. [9-11].

A 6-kDa early-secreted antigenic target (ESAT-6) is one of the most immunodominant Mycobacterium tuberculosis-specific antigen. This is because ESAT-6 contains multiple immunogenic T/B cell epitopes and, therefore, acts as a heterogeneous antigen tolerance break to induce an immune response [12, 13]. Interleukin (IL)-21 is a T cell-derived cytokine that regulates an immune response and generates considerable research interest in developing tumour vaccine as adjuvant molecule [11, 14]. Glycosylphosphatidylinositol (GPI) is a post-translationally added lipid anchor and GPI-anchored membrane cytokine has been shown to play an important role in host immune response against tumour cells [15, 16]. We had previously shown that administering the whole tumour cell vaccine expressing IL-21 in the GPI-anchored form induced protective antimelanoma immunity in a B16F10 cell transplantable mouse model. However, the antimelanoma efficacy did not fully inhibit tumour growth [4]. In the present study, we first developed a DNA vaccine ESAT-6-gpi and a modified melanoma vaccine B16F10-ESAT-6-gpi/IL-21 that could express heterogeneous antigen ESAT-6 and adjuvant molecule IL-21, respectively, and then adopted a novel immunization strategy of using the DNA vaccine ESAT-6-gpi priming and the tumour vaccine B16F10-ESAT-6-gpi/IL-21 boosting to further evaluate the antimelanoma effectiveness in the mice. Our data demonstrated that the novel immunization strategy elicited strong ESAT-specific immune responses that significantly inhibited melanoma growth in the immunized C57BL/6 mice. This is the first report that demonstrated the DNA vaccine ESAT-6-gpi as an immunomodulator for enhancing the tumour vaccine B16F10-ESAT-6-gpi/IL-21 against melanoma efficacy in the mouse model.

Materials and methods

Cells and animal

The B16F10 murine melanoma cell line is syngeneic in C57BL/6 mice; the S180 sarcoma cell line is a murine origin capable of growing in different inbred strains of mice. Both cell lines were obtained from the Cellular Institute of China in Shanghai. These cells were cultured at 37 °C in 5% CO2 atmosphere in RPMI 1640 supplemented with 10% fetal bovine serum that contained 100U/ml penicillin G sodium and 100 μg/ml streptomycin sulphate. C57BL/6 mice between 6 and 7 weeks of age were obtained from the Yangzhou University of China (licence number: SCXK, Jiangsu province of China, 2007–0001). All mice were housed under the pathogen-free condition and the experiments were performed in compliance with the guidelines of the Animal Research Ethics Board of Southeast University. Full details of approval of the study can be found in the approval ID: 20080925.

Development of DNA vaccine ESAT-6-gpi and tumour vaccine B16F10-ESAT-6- gpi/IL-21

The recombinants of pIRES-ESAT-6-gpi and pIRES-ESAT-6-gpi-IL-21 were constructed as described in our previous reports [13, 17]. The recombinants and mock plasmid were respectively transfected into the B16F10 cells using Lipofectamine TM 2000 reagent (Invitrogen, Carlsbad, CA, USA) by following the manufacturer's protocol. The stably-expressing ESAT-6-gpi cells and ESAT-6-gpi-IL-21 cells were respectively selected from RPMI containing 800 μg/ml G418 (Clontech, CA, USA) and were cloned by limiting dilution method. We named B16F10-ESAT-6-gpi/IL-21 cells for the cells expressing ESAT-6-gpi and IL-21; the B16F10-ESAT-6-gpi cells for the cells expressing ESAT-6-gpi; and the B16F10/mock cells for the cells with no expression. Expression of ESAT-6-gpi was detected by an immunofluorescence assay in the same way as described in our previous report [17]. Western blot was used to analyse the expression of IL-21 in the transfected cells. Briefly, 1 × 106cells were collected and lyzed in a protein extraction buffer (Novagen, WI, USA) by following the manufacturer's protocol. 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and proteins (10 μg/lane) were electrotransferred onto a nitrocellulose membrane. The goat anti-mouse IL-21 (I-18, Santa Cruz Biotechnology Company, Santa Cruz, CA, USA) was added to the membrane for 1 h, and the membrane was washed three times. The subsequent steps were performed by following to the Western Blot Kit's protocol (Pierce Company, Rockford, IL, USA) [18].

Animal experiment

In the antitumour experiment, the C57BL/6 mice were randomly divided into six groups: the phosphate-buffered saline (PBS) group; the B16F10/mock cell group; the B16F10-ESAT-6-gpi cell group; the pIRES-ESAT-6-gpi plus the B16F10-ESAT-6-gpi cell group; the B16F10-ESAT-6-gpi/IL-21 cell group and the pIRES-ESAT-6-gpi plus the B16F10-ESAT-6-gpi/IL-21 cell group. Each mouse was immunized subcutaneous (s.c.)/intramuscular (i.m.) with 50 μl PBS or 50 μl DNA vaccine compound containing 10 μg pIRES-ESAT-6-gpi, the 20 μl GenEscort™ III transfection reagent (Wisegen Biotech. Co. Ltd, Nanjing, China) and 20 μl PBS or 50 μl 1 × 106 B16F10/mock cells or 50 μl B16F10-ESAT-6-gpi cells or 50 μl B16F10-ESAT-6-gpi/IL-21 cells. The immunization was performed three times at a 10-day interval [19]. Table 1 shows the immunization schedule. Fig. 1 indicates the timeline of prime/boost immunizations. Finally, all mice were challenged s.c. in the flanks with 1 × 106 B16F10 cells 10 days after their final immunization. All the tumour cell vaccines were inactivated with 2 μg/ml mitoxantrone (MIT) before immunization. Twelve mice/group were used in the study. Tumour growth was monitored every 5 day by measuring two perpendicular tumour diameters using calipers, and then, the tumour volume, tumour-free mice and survival mice were evaluated, respectively.

Table 1. Mice immunization schedule*
GroupsPrimingBoosting
PBS50 μl PBS (i.m.)50 μl PBS × 3 (s.c.)
B16F10/mock cells50 μl PBS (i.m.)50 μl cells × 3 (s.c.)
B16F10-ESAT-6-gpi cells50 μl PBS (i.m.)50 μl cells × 3 (s.c.)
pIRES-ESAT-6-gpi + B16F10-ESAT-6-gpi cells50 μl DNA (i.m.)50 μl cells × 3 (s.c.)
B16F10-ESAT-6-gpi/IL-21 cells50 μl PBS (i.m.)50 μl cells × 3 (s.c.)
pIRES-ESAT-6-gpi + B16F10-ESAT-6-gpi/IL-21 cells50 μl DNA (i.m.)50 μl cells × 3 (s.c.)
Figure 1.

Timeline of prime/boost immunizations.

Preparation of dendritic cells (DCs) and detection of different molecular expression in the B16F10-ESAT-6-gpi/IL-21 tumour vaccine

The DCs from mouse bone marrow were prepared by following the protocol as described in reference [20]. The molecular expression of major histocompatibility complex (MHC) class II, the clusters of differentiation (CD)80 and CD11c was detected by following the manufacturer's protocol (eBioscience, California CA, USA) [21]. Briefly, the DCs or the tumour vaccine B16F10-ESAT-6-gpi/IL-21 cells were stained with the rabbit anti-mouse MHC class II-phycoerythrin (PE), CD 80-APC and CD11c-fluorescein isothiocyanate (FITC) monoclonal antibodies (eBioscience), respectively, and the subsequent steps were performed by following the Kit's protocol [22]. The tumour vaccine B16F10-ESAT-6-gpi/IL-21 cells inactivated with MIT were analysed using the AnnexinV-EGFP Apoptosis Detection Kit (KeyGen Biotech. Co. Ltd). The morphology of apoptotic cells was observed under a fluorescent microscope [TE2000-E fluorescence inverted phase contrast microscope (Nikon Corporation, Tokyo, Japan) ] [23].

Enzyme-linked immunospot (ELISPOT)/immunosorbent assay (ELISA)

Enzyme-linked immunospot assay for detecting IFN-γ was performed by following the Kit's protocol (eBioscience) [24, 25] Briefly, 96-well plates (Millipore) were coated with 15 μg/ml anti-IFN-γ mAb 1-DIK (Mabtech) at 4 °C overnight and blocked with 10% FCS for 1 h. Then, 1 × 105 splenocytes from the mice immunized with the different vaccines were added to wells in 100 μl volume and incubated with 2 × 104 B16F10-ESAT-6-gpi cells inactivated with MIT in 100 μl volume. After incubation for 24 h at 37 °C in 5% CO2, the cells were discarded, and the wells were washed five times with PBS containing 0.05% Tween 20 (Sigma-Aldrich). This was followed by incubation with 1 μg/ml biotinylated anti-IFN-γ mAb 7-B6-1-biotin (Mabtech) for 2 h at room temperature. Wells were washed again and incubated with streptavidin-conjugated alkaline phosphatase (Mabtech) for another 1 h. Individual cytokine-producing cells were identified as dark spots after a 30-min reaction with 5-bromo-4-chloro-3-indolyl phosphate and NBT by means of an alkaline phosphatase conjugate substrate kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Spots were counted using an automated reader (AID-Diagnostika Company, Strassberg, Germany), and results displayed as number of spot-forming cells (SFC) per 105 splenocytes. ELISA for detecting anti-ESAT-6 antibody was performed by following the Kit's protocol (eBioscience). The Kit is suitable for detection of samples including cell culture supernatant and serum, and the sensitivity of Kit is 5.3 pg/ml [26].

Complement-dependent cytotoxicity (CDC) and apoptotic tumour cells phagocytized by DCs

To detect CDC, we first plated 2 × 105 B16F10-ESAT-6-gpi cells or B16F10-Mock cells into a 96-well plate and then added immunized murine serum that contained anti-ESAT-6 antibody and the complement to the wells in different concentrations, respectively. The cells were incubated for 4 h, and the resulting absorption was analysed at 490 nm using a spectrophotometer. CDC was calculated by following the method as was described in the reference [27]. The collected DCs were incubated with the DiI-labelled B16F10-ESAT-6-gpi/IL-21 cells (red) and the immunized murine serum for 12 h. After washing, the precipitated cells were stained with FITC-conjugated anti-CD11c. The apoptotic tumour cells (red) phagocytized by DCs (green) were analysed using an immunofluorescence microscopic [20].

Assay of CD8+ T lymphocyte cytotoxicity

Ten days after the final immunization, 5 × 106 splenocytes were collected from the mice immunized with the DNA vaccine ESAT-6-GPI-prime and the tumour vaccine B16F10-ESAT-6-gpi/IL-21-boost. The CD8+ lymphocytes were isolated by the magnetic column selection or depletion (MACS, Miltenyi Biotec., Bergisch Gladbach, Germany) from the splenocytes [28, 29]. In the cytotoxic assay, the B16F10 cells and the S180 cells were used for target cells, respectively, and the harvested CD8+ lymphocytes were used for effector cells that were labelled with 0.5 mm 5-(and 6)-carboxy-fluorescein diacetate succinimidyl ester (CFSE; 20 μg/ml) at 37 °C for 15 min. The labelled CD8+ lymphocytes were washed twice in PBS containing 5% fetal bovine serum to sequester any free CFSE. The CFSE-labelled effector cells were seeded with a constant number of B16F10 target cells or S180 target cells in a 96-well plate at 10:1 and 25:1 ratios of effector cells to target cells (E:T). All the cytotoxicity assays were performed in triplicate. Flow cytometric CFSE/7-AAD cytotoxicity assays were analysed by Flow Cytometry (FCM, Becton, Dickinson and Company, San Jose, CA, USA) [17, 30].

Statistical analysis

The Student's t-test was used to evaluate the differences between the different experimental groups. Bonferroni correction was used where multiple comparisons were made. Differences at P < 0.05 were considered statistically significant.

Results

Target protein expression in the tumour vaccine B16F10-ESAT-6-gpi and B16F10-ESAT-6-gpi/IL-21

Figure 2A shows that the ESAT-6 was actually anchored on the surface of the B16F10-ESAT-6-gpi cells and the B16F10-ESAT-6-gpi/IL-21 cells, respectively, but not on the surface of the B16F10/mock cells. This is because the rat anti-mouse ESAT-6 was labelled with PE that exhibited red fluorescence under the fluorescence microscope and no red fluorescence was found on the B16F10/mock cells. IL-21 was correctly expressed in the B16F10-ESAT-6-gpi/IL-21 cells as is shown in Lane 3 in Fig. 2B. The results demonstrated that the IL-21 was expressed correctly in the B16F10-ESAT-6-gpi/IL-21 cells. To identify the IL-21 bioactivity in B16F10-ESAT-6-gpi/IL-21, the isolated CD8+lymphocytes from splenocytes in the normal mice were incubated with the supernatant from the cultured B16F10-ESAT-6-gpi/IL-21 and anti-CD3 antibody and the CD8+ lymphocyte's proliferative activity was significantly increased compared with the B16F10 cells (data not shown). The result suggested that the IL-21 was functional, and that these cells could be used further studying of the biological functions of the B16F10-ESAT-6-gpi and B16F10-ESAT-6-gpi/IL-21 tumour vaccines in the mouse model.

Figure 2.

Identification of target protein expression in the developed vaccines. The recombinants of pIRES-ESAT-6-gpi, pIRES-ESAT-6-gpi-IL-21 and mock plasmid were, respectively, transfected into the B16F10 cells. The cells with stable expressing of ESAT-6-gpi or ESAT-6-gpi-IL-21 were selected with G418. That the constructs expressed ESAT-6 and IL-21. (A) A strong PE fluorescence on the B16F10-ESAT-6-gpi cells and the B16F10-ESAT-6-gpi/IL-21 cells reflected that the ESAT-6 was successfully anchored on the B16F10 cell membrane via GPI. (B) That the IL-21 expression was in the B16F10-ESAT-6-gpi/IL-21 cells, but no IL-21 expression was in the B16F10/mock cells nor in the B16F10-ESAT-6-gpi cells.

Evaluation of DNA vaccine pIRES-ESAT-6-gpi enhancing tumour vaccine B16F10-ESAT-6-gpi/IL-21 efficacy against melanoma in mice

Figure 3A shows the following results: 2 of the 12 mice immunized with the DNA vaccine pIRES-ESAT-6-gpi plus the tumour vaccine B16F10-ESAT-6-gpi/IL-21 developed tumours on Day 25 and Day 30, respectively; 5 of the 12 mice immunized with the DNA vaccine pIRES-ESAT-6-gpi plus the tumour vaccine B16F10/ESAT-6-gpi developed tumours on Day 20, Day 25, Day 30, Day 35 and Day 40, respectively; 7 of the 12 mice immunized with the tumour vaccine B16F10-ESAT-6-gpi/IL-21 and 9 of the 12 mice immunized with the tumour vaccine B16F10/ESAT-6-gpi developed tumours, respectively. In addition, 10 of the 12 mice immunized with the B16F10-Mock vaccine developed tumours in 35 day; all 12 mice immunized with the PBS developed tumours in 20 days. Compared with the DNA vaccine B16F10-ESAT-6-gpi, the DNA vaccine pIRES-ESAT-6-gpi plus the tumour vaccine B16F10-ESAT-6-gpi and the tumour vaccine B16F10-ESAT-6-gpi/IL-21 also exhibited good effect against melanoma. However, the most powerful antimelanoma efficacy was found in the mice immunized with the DNA vaccine pIRES-ESAT-6-gpi plus the tumour vaccine B16F10-ESAT-6-gpi/IL-21, which was reflected in 10 of the 12 mice in this group were tumour-free until 60 days into the observation (Fig. 3B).

Figure 3.

DNA vaccine ESAT-6-gpi-prime and tumour vaccine B16F10-ESAT-6-gpi/IL-21 boost-augmented antimelanoma efficacy in mice. The tumour volume (A) and the tumour-free mice (B) in the C57BL/6 mice immunized with the different tumour vaccines. The experimental schedule of immunization of mice is shown as in Table 1 and Fig. 1. The results indicated that B16F10-ESAT-6-gpi vaccine did not significantly inhibit tumour growth compared with B16F10-Moke, but the addition of IL-21 in the vaccine did inhibit tumour growth. Clearly, the strongest antimelanoma efficacy was found in the DNA vaccine ESAT-6-gpi-prime and the tumour vaccine B16F10-ESAT-6-gpi/IL-21 boost group. 12/12 represents 12 of the 12 mice generated tumour. The others were analogized. *P < 0.05; ns: no statistical significance.

Induction of immune responses in mice immunized with the DNA vaccine ESAT-6-gpi

To analyse the mechanisms of the DNA vaccine ESAT-6-gpi for reinforcing the antimelanoma efficacy of the tumour vaccine B16F10-ESAT-6-gpi/IL-21, we firstly detected immune responses in mice induced by the DNA vaccine ESAT-6-gpi. Fig. 4A indicates that the serum anti-ESAT-6 antibody was significantly increased in the DNA vaccine pIRES-ESAT-6-gpi group compared with the pIRES-mock plasmid group (< 0.005). The serum anti-ESAT-6 antibody was bound to the ESAT-6-gpi on the surface of the B16F10-ESAT-6-gpi cells (the dotted line in Fig. 4B). The CDC in the DNA vaccine ESAT-6-gpi group was significant higher (36.72%) than that of the pIRES mock plasmid group (8.96%) in Fig. 4C. The statistical analysis is shown in Fig. 4D (< 0.01). Fig. 4E shows that the serum IFN-γ level in the mice immunized with the DNA vaccine ESAT-6-gpi was significantly increased in contrast to that in the mice immunized with the pIRES mock plasmid (70 ± .8.9 versus 32 ± .3.1, < 0.01). High cytotoxicity was significantly found in the DNA vaccine ESAT-6-gpi group, at a 25:1 ratio of CD8+ effector cells to B16F10 target cells (Fig. 4F–G, 32.21% versus 14.33%, < 0.005). These results suggested that the DNA vaccine ESAT-6-gpi elicited the humoral and cellular immune responses, which might assist in reinforcing the antitumour immune efficacy of the tumour vaccine B16F10-ESAT-6-gpi/IL-21 boosting in mice.

Figure 4.

Detection of immune responses induced by the DNA vaccine ESAT-6-gpi. (A) The serum anti-ESAT-6 antibody in the mice immunized with the mock plasmid or the DNA vaccine pIRES-ESAT-6-gpi detected by ELISA. Serum anti-ESAT-6 antibody bound ESAT-6-gpi on the surface of B16F10-ESAT-6-gpi cells (the right dotted line) detected by FCM (B). A complement-dependent cytotoxicity (CDC) was detected with the serum that contains anti-ESAT-6 antibody and the complement (C). (D) The statistical result of CDC between the B16F10-Mock cells and the B16F10-ESAT-6-gpi cells. The serum IFN-γ level in the immunized mice was detected by ELISA (E). The cytotoxicity of CD8+ T lymphocytes to B16F10 target cells at 25:1 ratio or 10:1 ratio was detected by FCM in (F) and the statistical analysis of cytotoxicity is shown in (G). *P < 0.05, **P < 0.01 and ***P < 0.005.

Analysis of the mechanisms of strengthening antimelanoma efficacy by the DNA vaccine ESAT-6-gpi priming and the tumour vaccine B16F10-ESAT-6-gpi/IL-21 boosting

After we found that the DNA vaccine ESAT-6-gpi was capable of enhancing the antimelanoma efficacy of the tumour vaccine B16F10-ESAT-6-gpi/IL-21 (Fig. 3) and the immune efficacy in mice (Fig. 4), we attempted to know whether this efficacy could induce the strong CD8+ T cell response that was specific to the heterogeneous antigen ESAT-6 expressed in the B16F10 tumour cells. In addition, we hoped to understand whether this efficacy was associated with the maturation of DCs that phagocytized the apoptotic tumour cells and expressed immune molecules. We found that punctiform or cluster ESAT-6-gpi with the red fluorescence was distributed on the surface of the apoptotic tumour cells (arrow, Fig. 5A) and the apoptotic bodies (arrow, Fig. 5B) appeared in the tumour vaccine B16F10-ESAT-6-gpi/IL-21. We hypothesized that the serum anti-ESAT-6 antibody was bound to ESAT-6 on the surface of the apoptotic tumour cells to form the apoptotic tumour cell-immune complex (ATC-IC) that might promote DCs to phagocytize ATC-IC. Figure 5C indicates the gating strategy used to gate out the DCs. From the FCM results, we found that the percentage of ATC-IC phagocytized by DCs was gradually increased after the different tumour cells were incubated with the anti-ESAT-6 serum and DCs (Fig. 5D–J). For examples, the percentage of DC phagocytized ATC-IC in the tumour vaccine B16F10/ESAT-6-gpi cells plus anti-ESAT-6 serum antibody (60.62%, Fig. 5H) was remarkably elevated in contrast to that of the tumour vaccine B16F10-ESAT-6-gpi cells alone in Fig. 5G (52.86%, P < 0.05), and this is also true of the tumour vaccine B16F10-ESAT-6-gpi/IL-21 cells plus serum anti-ESAT-6 antibody (65.01%, Fig. 5J) in contrast to the tumour vaccine B16F10-ESAT-6-gpi/IL-21 cells alone in Fig. 5I (59.54%, P < 0.05). The B16F10 cells plus anti-ESAT-6 serum in Fig. 5F was, however, not enhanced much (49.53%) compared with the B16F10 cells in Fig. 5E (47.09%, P > 0.05). Figure 5D served as a negative control. Figure 5K reveals that the image of ATC-IC (red) phagocytized by DCs (green) was seen under the fluorescence microscope. The results suggested that the serum anti-ESAT-6 antibody might have promoted the DC maturation after the DC phagocytized ATC-IC (Fig. 5L). The DC maturation was reflected in the enhanced the molecular expression of MHC class II and CD 80 on the surface of DCs from the mice immunized with the tumour vaccine B16F10-ESAT-6-gpi cells plus serum anti-ESAT-6 antibody as well as the tumour vaccine B16F10-ESAT-6-gpi/IL-21 cells plus serum anti-ESAT-6 antibody (Fig. 5M).

Figure 5.

Apoptotic tumour cell-immune complex (ATC-IC) promoted DCs to phagocytize an apoptotic bodies and to be maturation. (A,B) Indicate the ESAT-6-gpi on the apoptotic tumour cells and apoptotic bodies in tumour cells under an immunofluorescence microscope. The arrows show the ESAT-6-gpi (white dashed line) and the apoptotic bodies (yellow solid line) that were originated from the tumour vaccine B16F10-ESAT-6-gpi/IL-21 cells inactivated with mitomycin for 48 h. (C) The gating strategy used to gate out the DCs. (D–J) Represent that the B16F10-Mock cells, the B16F10 cells plus anti-ESAT-6 serum, the B16F10-ESAT-6-gpi cells, the B16F10-ESAT-6-gpi cells plus serum anti-ESAT-6 antibody, the B16F10-ESAT-6-gpi/IL-21 cells, the B16F10-ESAT-6-gpi/IL-21 cells plus serum anti-ESAT-6 antibody and the control B16F10 cells with no any treatment in order and that exhibit the percentage of DC phagocytosis of ATC-IC, relative to a negative culture medium control, detected by FCM 12 h after the different apoptotic tumour cells were incubated with DCs and serum anti-ESAT-6 antibody. The collected DCs were used for incubation together with the DiI-labelled B16F10-ESAT-6-gpi/IL-21 cells (red) and the serum anti-ESAT-6 antibody for 12 h and then stained with FITC-conjugated anti-CD11c. An apoptotic tumour cell phagocytized by DCs (green) was observed under immunofluorescence microscopic as shown in (K,L) exhibits the ratio of DC phagocytized ATC-IC. The expression of MHC class II and CD80 molecules on the surface of DCs detected by FCM (M) *P < 0.05 and **P < 0.01. 01. The percentage of DC phagocytosis of ATC-IC was detected by FCM after the different apoptotic tumour cells were incubated with DCs and serum anti-ESAT-6 antibody 12 h, and the results are shown in (D–J) which represent the B16F10-Mock cells, the B16F10 cells plus anti-ESAT-6 serum, the B16F10-ESAT-6-gpi cells, the B16F10-ESAT-6-gpi cells plus serum anti-ESAT-6 antibody, the B16F10-ESAT-6-gpi/IL-21 cells, the B16F10-ESAT-6-gpi/IL-21 cells plus serum anti-ESAT-6 antibody and the control B16F10 cells with no treatment in order.

The results given in Fig. 6A indicate that the activity of CD8+CTL to the B16F10 cells was significantly increased in the 25:1 ratio of effector cells to target cells after the third immunization, especially in the group with DNA vaccine ESAT-6-gpi plus the tumour vaccine B16F10-ESAT-6-gpi/IL-21 (Fig. 6B). However, no remarkable cytotoxicity to the S180 cells was found in the two groups (Fig. 6C and Fig. 6D). This was mainly because that the CD8+CTL activity was only specific to B16F10 target cells but not to the S180 cells in the mice immunized with the B16F10-ESAT-6-gpi cells or the B16F10-ESAT-6-gpi/IL-21 cells. In Fig. 6E, the highest number of IFN-γ-SFC was found in the mice immunized with DNA vaccine ESAT-6-gpi plus the tumour vaccine B16F10-ESAT-6-pi/IL-21. These results demonstrated that priming with the DNA vaccine ESAT-6-gpi in combination with boosting with the tumour vaccine B16F10/ESAT-6-gpi did elicit strong immune responses that might induce powerful antitumour efficacy in the immunized mice.

Figure 6.

Detection of CD8+ CTL activities and IFN-γ-producing lymphocyte numbers. (A) Indicates the activities of CD8+ CTL in the mice immunized with the different vaccines. 10:1 and 25:1 represent the ratios of the effector cells (CD8+ lymphocytes isolated by MACS from the splenocytes in the immunized mice) to the target cells (B16F10 cells). (B) The statistical analysis of cytotoxicity. (C) Suggests that the CD8+ CTL did not kill the S180 cells at the 25:1 ratio of effector cells to target cells in the mice immunized with the two vaccines and there is no statistical significance between this two vaccines (D,E) The number of IFN-γ-spot-forming cells (SFC) per 105 splenocytes. *P < 0.05.

Discussion

Melanoma is a mutated ‘self’, which makes the host immune system essentially tolerates in the absence of any external perturbation. A major obstacle to the success of any form of specific tumour vaccine immunotherapy might be the tumours that escape from immune recognition. In this study, we used the Mycobacterium tuberculosis heterogeneous antigen ESAT-6 anchored by the GPI in our engineered the DNA vaccine ESAT-6-gpi and the tumour vaccine B16F10-ESAT-6-gpi/IL-21, respectively, to break immune tolerance in melanoma bearing mice and to augment the efficacy of antimelanoma immunity through DNA vaccine ESAT-6-gpi-prime and tumour vaccine B16F10-ESAT-6-gpi/IL-21-boost in a mouse model.

Our findings from the study showed that this innovative approach did elicit a strong antimelanoma immunity to inhibit melanomatous growth after being challenged with the B16F10 cells in the mouse model. Though the DNA vaccine ESAT-6-gpi priming and the tumour vaccine B16F10-ESAT-6-gpi boosting or the tumour vaccine B16F10-ESAT-6-gpi/IL-21 alone showed obvious efficacy against melanoma as well, this efficacy was more efficient in the mice immunized with the DNA vaccine ESAT-6-gpi-prime and the tumour vaccine B16F10-ESAT-6-gpi/IL-21 boost (Fig. 3). Our results from the antimelanoma experiments also suggested that although the DNA vaccine ESAT-6-gpi or the DNA vaccine ESAT-6-gpi-prime and the tumour vaccine B16F10-ESAT-6-gpi boost could break immune tolerance in melanoma bearing mice [16, 31, 32], the antimelanoma efficacy of these vaccines was not sufficiently strong. This lack of sufficient strength was due to the absence of molecular adjuvant IL-21 that would induce strong antitumour immunity in the tumour vaccine B16F10-ESAT-6-gpi/IL-21 [4, 11, 26].

To analyse the probable mechanisms of the DNA vaccine ESAT-6-gpi to augment the tumour vaccine B16F10-ESAT-6-gpi/IL-21 antimelanoma efficacy in the mouse model, we first investigated the immune responses induced by the DNA vaccine that had emerged as a potentially important form of antigen-specific immunotherapy [32, 33]. Because the Mycobacterium tuberculosis ESAT-6 attribute is of primary importance in tuberculosis immunodiagnosis and vaccine development [12, 32, 34, 35], we therefore hypothesized that the ESAT-6-gpi might be easily recognized by immune cells in the mice immunized with both the DNA vaccine ESAT-6-gpi and the tumour vaccine B16F10-ESAT-6-gpi/IL-21. The serum anti-ESAT-6 antibody bound ESAT-6 on the surface of tumour vaccine B16F10-ESAT-6-gpi/IL-21 cells to form immune complex that subsequently activated the complement classical pathway and resulted in CDC. The anti-ESAT-6 antibody in the immunized serum, we also guess, might have activated natural killer (NK) cells after serum anti-ESAT-6 antibody bound the Fc γ receptor of NK cells through the Fc fragment, which resulted in antibody-dependent cell cytotoxicity (ADCC) (not investigated in this study). The tumour vaccine B16F10-ESAT-6-gpi/IL-21 cells, therefore, could be killed by both CDC and ADCC. Apoptotic B16F10-ESAT-6-gpi/IL-21 tumour cells served as a potential potent trigger for tolerance break to elicit an initiation of naturally occurring melanoma immunity because apoptotic tumour cells might be an excellent source for DC loading when apoptotic tumour cells bound specific serum antibody to form ATC-IC. The potential uncharacterized antigens of ATC-IC would be efficiently presented to CD8+ T lymphocytes without any prior characterization and isolation of these antigens [36]. This was revealed by the augmented DC phagocytosis of ATC-IC through the immune opsonization that facilitated DC maturation (Fig. 5). And, these were also revealed by the elevated serum levels of anti-ESAT-6 antibody and IFN-γ and enhanced the cytotoxicities of CDC and CD8+CTL as well as the increased IFN-γ-spot-forming cell numbers. The enhanced immune effect resulted in augmenting antimelanoma efficacy in the mice immunized with the DNA vaccine ESAT-6-gpi priming and the tumour vaccine B16F10-ESAT-6- gpi/IL-21 boosting. In the current study, the DNA vaccine ESAT-6-gpi was found to be an effective vaccine for enhancing the potency of the tumour vaccine B16F10-ESAT-6-gpi/IL-21, of which the heterogeneous antigen ESAT-6 broke the immune tolerance in melanoma and induced a potent immune responses in the mouse model, while IL-21 acted as a tumour vaccine adjuvant to augment the murine antitumour efficacy [11, 37]. This is because IL-21 could induce secondary cytokine production, particularly IFNγ that was generated by CTLs and NK cells. Consequently, IFNγ again reacted on NK cells and CTLs, enhancing their cytotoxic activities, playing a pivotal biological role in killing tumour cells or inducing tumour cell apoptosis [4, 11, 22].

In conclusion, our data demonstrated that the DNA vaccine ESAT-6-gpi priming and the tumour vaccine B16F10-ESAT-6-gpi/IL-21 boosting stimulated the generation of strong antitumour immunity in mice, which inhibited melanomatous growth in the mouse model. Therefore, using the DNA vaccine ESAT-6-gpi-prime might be a promising strategy for increasing the potency of tumour vaccine B16F10-ESAT-6-gpi/IL-21 boost in prophylactic vaccination against murine melanoma.

Acknowledgment

This work was partly supported by the National Natural Science Foundation of China (No. 81071769, No. 81202372) and part by the 973 National Nature Science Foundation of People's Republic of China (2011CB933500).

Conflict of interest

The authors declare no conflict of interest.

Ancillary