Dendritic cells modified by tumor associated antigen SMP30 have enhanced antitumor effect against mouse hepatocarcinoma cells in vitro and in vivo

Abstract

Objectives: Tumor immunotherapy based on dendritic cells (DC) is one of the most promising approaches to treat cancers. This therapy uses an immunogenic tumor antigen to present it to T cells. Senescence marker protein 30 (SMP30) is identified as a tumor associated antigen (TAA) with high immunogenicity and specificity for hepatocellular carcinoma (HCC). DCs are the most potent antigen presenting cells, and can be transduced with tumor antigens to enhance antitumor immune response. The purpose of this study was to investigate the antitumor effect of DCs transduced with a recombinant lentiviral vector (LV-SMP30) expressing SMP30. Methods: A recombinant lentiviral vector (LV-SMP30) expressing SMP30 was constructed and transduced into DCs. The expression of SMP30 was detected by western blot. Mouse bone marrow-derived DCs were divided into four groups: LV-SMP30 group (transduced with LV-SMP30), Protein group (co-cultured with SMP30 protein), LV group (transduced with the empty vector) and Untreated group (the normal DCs). The effect of LV-SMP30 on DCs was detected through surface markers (CD123, CD11c, CD80 and CD86) and cytokine production. The activation and proliferation of CD3⁺CD8⁺ T cells were detected by CCK-8 kit. Flow cytometry was used to detect CD3⁺CD8⁺ T cell-mediated cytotoxicity. After construction of a mouse subcutaneous xenograft model, the volume and growth of tumors in different groups were observed. The changes in serum immune indexes in the treated groups were compared with those in the control group. Results: The LV-SMP30 recombinant was constructed and transduced into DCs successfully, and LV-SMP30-transduced DCs stably expressed SMP30. The percentages of expression in the LV-SMP30 and Protein groups were significantly higher than those in the LV or Untreated groups (P<0.05). Meanwhile, after the DCs were cultured for 72 hours, the levels of IL-2, IL-6, IL-12, and IFN-γ were significantly higher in the LV-SMP30 and Protein groups than in the LV group or Untreated group (P<0.05). After the DCs were continuously cultured for one week, however, the cytokine levels in the LV-SMP30 group were significantly higher than those in the Protein group (P<0.05). In addition, CD3⁺CD8⁺ T cell proliferation and activation levels were substantially higher in the LV-SMP30 and Protein groups than in the LV or Untreated groups (P<0.05). Furthermore, as the ratio of effectors/target cells increasing in the LV-SMP30 group, CD3⁺CD8⁺ T cell-mediated cytotoxicity in H22 cells became higher (0:1, 10:1; 20:1; 40:1, respectively). In comparison to the control group, the cytotoxicity of the LV-SMP30 group was considerably increased at the ratios of 10:1, 20:1 and 40:1 (P<0.05). However, in the case of Hep1-6 cells, there was no significant difference in CD3⁺CD8⁺ T cell-mediated cytotoxicity among the groups. In addition, when compared with other groups, the mice in the LV-SMP30 group showed the most volume reduction, the slowest tumor growth, and the highest level of IL-2 and IFN-γ (P<0.05). Conclusion: DCs transduced with LV-SMP30 can dramatically enhance specific CD3⁺CD8⁺ T cell immune responses against mouse hepatocarcinoma cells in vitro and in vivo. These findings lend significant support to the development of the DC-based SMP30 antigen vaccine for hepatocarcinoma immunotherapy.

Introduction

Vaccination based on dendritic cells (DCs) is one of the most promising cancer immunotherapy techniques. Many studies have demonstrated that a DC-based vaccination can elicit an efficient antitumor immune response [1,2]. The primary basis for the immune system to eliminate tumors is the existence of immunogenic tumor antigens recognized by B and T cells [3]. Therefore, screening and identifying appropriate tumor antigens is a precondition for cancer immunotherapy.

In the past few years, more than 2000 tumor antigens, including NY-ESO-1 and MAGE, have been identified by SEREX (serological identification of antigens by recombinant expression cloning) [4,5]. We previously used SEREX to screen and identify a range of tumor associated antigens, including Senescence marker protein 30 (SMP30), GC-binding factor 2 (GCF2) and Kinectin, from Guangxi human hepatocellular carcinoma (HCC) cDNA library [6,7]. SMP30 was overexpressed in most human and animal liver cancer tissues and cells, and it was strongly linked to liver cancer development and poor clinical outcome [8,9]. Most importantly, SMP30 is highly immunogenic and specific for HCC [10,11]. Hence, SMP30 appears to be a suitable tumor antigen for HCC immunotherapy.

DCs are the most potent antigen-presenting cells according to previous research [12]. DC activation is linked to antigen acquisition and cytokine production [3]. DCs are typically loaded by incubation with tumor peptides, tumor lysates or tumor cells to improve the activation as well as cytotoxicity against cancer [3,13-15]. DCs pulsed with tumor antigen peptides can elicit a T cell antitumor immune response in vitro [16,17]. After co-incubation with SMP30 peptide, DCs were activated and then induced the naïve T cell activation, and our previous study showed that the latter mediated specific cytotoxic T cell immune response against HCC cells [10,11,18]. However, the limitations of SMP30 peptide, including unstable expression and degradation of SMP30, severely limited its induction of DCs.

Gene modification is an important strategy in gene function research that can overcome the limitation of tumor antigen peptides [19]. In terms of current technology, DCs can be modified in a variety of ways, including gene insertion encoding tumor antigen. Gene insertion can offer multiple antigen epitopes to DCs while increasing immune duration [19,20]. Lentiviral vector (LV) is another effective tool that is widely used in gene function research. LV can integrate antigenic genes into the target cell genome and sequentially produce a continuous expression of tumor antigen in target cells [19,20]. Those target cells include dividing and non-dividing cells, such as T cells and DCs [3,21,22].

In the current study, we first constructed a SMP30 gene expressed recombinant LV (LVSMP30) and sequentially transduced it into DCs derived from mouse bone marrow. We investigated whether LV-SMP30 could increase DC activation and proliferation, as well as whether LV-SMP30-transduced DCs could enhance CD3⁺CD8⁺ T cell-mediated antitumor immune response. Our results revealed that LV-SMP30 significantly increased DC activation and proliferation, and LV-SMP30-transduced DCs intensively enhanced CD3⁺CD8⁺ T cell-mediated cytotoxicity against specific liver cancer cells in mice.

In addition, we successfully constructed a BALB/C nude mouse model to further verify the CD3⁺CD8⁺ T cell-mediated cytotoxicity. In subcutaneous xenograft models, we also assessed whether LV-SMP30-transduced DCs could elicite CD3⁺CD8⁺ T cell-mediated antitumor immune response. Our findings revealed that the LV-SMP30 group showed a better anti-tumor effect in vivo than the other groups. All these findings encourage the development of a DC-based SMP30 antigen vaccines for future HCC immunotherapy.

Materials and methods

Cell culture

Mouse liver cancer cell lines H22 and Hep1-6 were employed, purchased from iCell Bioscience Inc. In a humidified 5% CO₂ incubator at 37°C, the cells were cultured in RPMI-1640 media (Hyclone, UT, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA), 200 U/ml penicillin and 100 mg/ml streptomycin.

Animals

BALB/c mice (6-8 weeks old) and BALB/c nude mice (4-6 weeks old) were purchased from Beijing Huafukang Biotechnology, China, and kept at the animal experiment center of Guangxi Medical University. The housing conditions were an SPF level environment with 4-6 mice per cage with a laboratory temperature of 20°C-25°C and a humidity of 50%. All animal breeding was in compliance with the principles and procedures prescribed by the Animal Management and Use Committee of Guangxi Medical University.

Construction of recombinant lentivirus vector (LV-SMP30) expressing SMP30

RT-PCR was used to amplify the SMP30 gene from mouse cells. The following primers were used for PCR amplification of a 900 bp-long SMP30 gene: 5’-AGG GTT CCA AGC TTA AGC GGC CGC GCC ACC (forward); 5’-GAT CCA TCC CTA GGT AGA TGC ATT CAC CCT GCA TAG GAA TAT GGG GCA ATT CCT TTG ACT-3’ (reverse). The PCR product was inserted into NotI sites of LV5 Shuttle Plasmid (Genepharma, Suzhou, China). LV5 Shuttle Plasmid encoded a Green Fluorescent Protein (GFP) to track transduction efficiency. Direct sequencing was used to confirm the DNA sequence of the positive clone.

Preparation of DCs and transduction with LV-SMP30

Mouse DCs (i.e., mononuclear cells derived from bone marrow of BALB/c mice) were donated by Guangxi Key Laboratory of Biological Targeted Diagnosis and Treatment. DCs were incubated in RPMI 1640 medium with 10% FBS (Gibco) for 3-4 hours. Then, they were adhered to the wall by inducing granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 7 days and stimulated with Lipopolysaccharide (LPS) for 24 hours.

After that, the DCs were transduced with LV-SMP30. On the day before infection, DCs were seeded in 24-well plates at a density of 0.5 × 10⁵ cells per well with 0.5 ml of complete culture media overnight at 37°C with 5% CO₂. After 12-24 hours, DCs were cultured overnight at 37°C in a 5% CO₂ incubator using RPMI1640 media supplemented with 10% FBS, 0.5 ml of 10-fold diluted lentivirus solution and polybrene infection promoter at a final concentration of 5 μg/ml. The supernatants were discarded 24 hours after infection, and DCs were incubated in fresh medium for another 72 hours. GFP expression was observed using a fluorescence microscope to assess efficiency. In this study, the group of DCs transduced with LV-SMP30 was named as LV-SMP30 group. The group with positive control of DCs co-cultured with SMP30 protein (100 ng/ml) was named the Protein group. The control groups were designed as follows as a parallel control: DCs transduced with the empty vector (named as LV group) and the normal DCs (named as Untreated group).

Western blot assay for measuring SMP30 protein expression

Total proteins were extracted from DCs in each group using RIPA buffer (Solarbio, Beijing, China), separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a PVDF membrane. After incubation with 5% nonfat milk for 2 hours at room temperature, the membrane was treated with anti-SMP30 antibody (Fitzgerald, USA). After being washed, the membranes were incubated with an alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (Beyotime, Shanghai, China) for one hour at room temperature. Electrochemiluminescence (ECL) was used to detect immunoreactive bands. The manufacturer’s instructions were followed during the western blot assay.

Flow cytometry (FCM) analysis for determining LV-SMP30-transduced DC surface markers

DC surface staining utilized direct immunofluorescence and was analyzed by FCM (BD/Biometra). The following fluorescent mAbs were used to identify surface markers: anti-CD123 (5B11), anti-CD11c (N418), anti-CD8a (53-6.7), anti-CD80 (1G10), anti-CD86 (GL1) and anti-CD3 (17A2). All of the Abs mentioned above were purchased from eBioscience Inc. To analyze the phenotypes of the LV-SMP30-transduced DCs, 1 × 10⁶ DCs per tube were collected and the supernatant was removed after centrifuging. Then, the cells were washed once with precooled PBS and incubated with the fluorescent mAbs (CD123, CD11c, CD80, CD86, CD3 and CD8a, respectively) for 30 minutes at room temperature in the dark. FCM was used to perform fluorescence-activated cell sorting and analysis after the cells were washed and resuspended in PBS.

Determination of cytokines by ELISA

ELISA kits (Cusabio, Wuhan, China) were used to measure the concentrations of IL-2, IL-6, IL-12 and interferon-γ (IFN-γ) in the supernatant of culture media according to the manufacturer’s instructions. DCs were cultured for 72 hours and 7 days in 24-well plates, then adjusted to 5 × 10⁵ cells/ml. After 48 hours, the supernatant from each group was collected to measure IL-12 and IFN-γ. Likewise, the concentrations of IL-2, IL-6, IL-12, and IFN-γ in the supernatant of DCs co-cultured with mice peripheral blood mononuclear cells (PBMC) for 72 hours and 7 days were also measured.

Isolation and purification of CD3⁺CD8⁺ T cells

PBMCs were first isolated from six BALB/c mice (6-8 weeks old) using a lymphocyte isolation method. Then, PBMCs were induced for 2 days with 500 U/ml IL-2 (Novartis, Camberley, UK), 0.5 µg/ml anti-CD3 (BD Pharmingen, Cowley, UK) and 0.5 µg/ml anti-CD28 (BD Pharmingen, Cowley, UK). Following that, PBMCs weredifferentiated into T cells using only IL-2 to maintain cell culture for 7 days. Finally, CD8⁺ T cells were isolated using CD8⁺ T magnetic beads separation kit (Miltenyi Biotech, Gladbach, Germany) according to the manufacturer’s protocol. The purity of CD3⁺CD8⁺ T cells was examined by FCM.

Detection of CD3⁺CD8⁺ T cells proliferation by CCK-8

DCs were co-cultured with mice peripheral blood mononuclear cells (PBMC). After acquiring DC-presented antigens, the proliferation of naive T cells during the activation of CD3⁺CD8⁺ T cells was detected by CCK-8 kit (Abcam, Shanghai, China) according to manufacturer’s protocol.

Detection of CD3⁺CD8⁺ T cell-mediated cytotoxicity in vitro by FCM

CD3⁺CD8⁺ T cell-mediated cytotoxicity was measured by FCM and the procedure was performed as before [23]. Briefly, H22 cells and Hep1-6 cells were labeled with green fluorescent dye DIOC18 (Beyotime, Shanghai, China) before co-incubation with CD3⁺CD8⁺ T cells. After co-incubation, the red fluorescent PI (Beyotime, Shanghai, China) was employed to mark dead cells. The cell-mediated cytotoxicity was expressed as percentage. The formula is as follows: Cytotoxicity = [Control-viable cell %]-[Co-incubation-viable cell %]/[Control-viable cell %] [23].

Construction of BALB/c nude mice model

BALB/c nude mice (4-6 weeks old) were divided into 4 groups. H22 cells (1 × 10⁷ H22 cells, 0.1 ml) were injected subcutaneously into the right abdomen of mice. On approximately the seventh day, when the tumor had grown to approximately 100 mm³, the model was deemed successful, and the mice were randomly assigned to 4 groups: LV-SMP30 group, Protein group, LV group, Untreated group and PBS group. Then, 0.3 ml bulk CD3⁺CD8⁺ T cells (3 × 10⁷ CD3⁺CD8⁺ T cells, 0.1 ml) were injected from the caudal vein every four days until the 13th day, and the mice were sacrificed on the 15th day after vaccination.

Recording the changes in tumor volume for each group

Tumor size was assessed with a caliper before CD3⁺CD8⁺ T cells were injected into BALB/c nude mice in each group. Tumor size was measured every 3 days after CD3⁺CD8⁺ T cell injection until the mice were euthanized, and tumor volume was calculated.

Calculation of tumor inhibition rate

The mice in each group were euthanized on the 15th day after CD3⁺CD8⁺ T cell injection, and then the tumor tissues were completely stripped and weighed. Tumor inhibition rate was calculated according to the following formula. Tumor inhibition rate (%) = (average tumor weight of PBS group-average tumor weight of the experimental group)/average tumor weight of PBS group * 100%.

Measurement of cytokines by ELISA

On the 15th day following injection of CD3⁺CD8⁺ T cells, the levels of cytokine INF-γ and IL-2 in the serum of mice in each group were measured by ELISA kit (Cusabio, Wuhan, China) according to the manufacturer’s instructions.

Statistical analysis

Statistical analysis used Statistica 7.0 (Dell, Austin, TX, USA). All data are presented as the Mean ± SD. The t-test was used to evaluate statistical significance for two-group comparisons, and one-way analysis of variance (ANOVA) was used for three-group comparisons. Statistical significance was defined as P<0.05.

Results

Construction of the recombinant LV-SMP30 expressing SMP30

To construct the recombinant LV-SMP30 expressing SMP30, the 900 bp-length SPM30 gene derived from mice was amplified, purified and cloned into NotI sites of a LV5 (Figure 1). The LV5 containing SMP30 insertion was confirmed by direct sequencing.

Figure 1.

Construction of recombinant LV5 expressing SMP30 gene.

Stable expression of SMP30 protein in DCs transfected with LV-SMP30

Mouse DCs were transfected with LV-SMP30. GFP expression was used to test the transduction efficiency of DCs under a fluorescence microscope (Figure 2A). Transduction efficiency in the LV-SMP30 group was nearly 70% 72 hours after transfection.

Figure 2.

DCs were transfected with LV-SMP30 or LV. A. GFP expression was used to assess transduction efficiency under a fluorescence microscope (× 200). 72 hours after transfection, the transduction efficiency in the LV-SMP30 group was nearly 70%. B. Western blot was used to examine SMP30 protein expression. The results showed that SMP30 protein increased more significantly in the LV-SMP30 group than in the LV-group or Untreated group. Overall, the results confirmed that LV-SMP30 was an effective recombinant that could express SMP30 protein in a stable manner.

SMP30 protein was examined in DCs by western blot to establish the ability of LV-SMP30 on protein expression. The results revealed that SMP30 protein increased more significantly in the LV-SMP30 group than in the LV group or Untreated group (Figure 2B). The results suggested that LV-SMP30 is an effective recombinant that can express SMP30 protein in a stable manner.

Surface marker identification of LV-SMP30-transduced DCs

CD123 and CD11c are commonly employed to identify markers of DCs [3], while the upregulation of CD80 and CD86 are used as the activation markers of DCs [3]. As illustrated in Figure 3A, CD123 had a substantially larger cell percentage than CD11c [(64.9±0.06)% vs. (0.29±0.04)%] (P<0.05), indicating that DC123 was the main subtype of DCs in mice. Additionally, the cell percentages of CD80 and CD86 were (86.3±0.30)% and (72±13.12)%, respectively, in the LV-SMP30 group. Both percentages were considerably higher in the LV-SMP30 group than in the LV group or Untreated group (P<0.05) (Figure 3B, 3C). The results above suggested that LV-SMP30 enhanced DC activation.

Figure 3.

Phenotype identification of DCs by surface markers. A. The percentage of CD123 was substantially higher in comparison to CD11c [(64.9±0.06)% vs. (0.29±0.04)%, P<0.05], indicating that DC123 was the main type of DCs in mice. B. The cell percentage of CD80 increased more significantly in the LV-SMP30 group and Protein group than in the LV group or Untreated group (P<0.05). C. Similarly, the cell percentage of CD86 increased more significantly than in the LV group or Untreated group (P<0.05). Overall, the results indicated that LV-SMP30 improved DC maturation. A bar graph shows statistical significance for group comparisons.

LV-SMP30-transduced DCs induce specific cytokine production

Cytokines released during DC activation can strongly influence the immune responses through IL-2, IL-6, IL-12 and INF-γ [3,4]. These cytokines are mainly secreted by Th1 cells. After 72 hours of culture of DCs, the levels of IL-2, IL-6, IL-12, and IFN-γ in the LV-SMP30 group and Protein group were substantially higher than those of the LV group or Untreated group (P<0.05) (Figure 4A). After one week of continuous culture of DCs, the levels of IL-2, IL-6, IL-12, and IFN-γ in the LV-SMP30 group were significantly higher than those of the Protein group (P<0.05) (Figure 4A).

Figure 4.

IL-2, IL-6, IL-12 and IFN-γ concentrations in supernatant were analyzed by ELISA. A. At 72 hours, IL-2, IL-6, IL-12 and IFN-γ in LV-SMP30 group and Protein group were increased significantly in comparison with those of the LV-group or Untreated group (P<0.05). At one week, the levels of IL-2, IL-6, IL-12 and INF-γ in the LV-SMP30 group were significantly higher than those in the Protein group (P<0.05). The results suggest that LV-SMP30 possesses a strong capability to induce the production of IL-2, IL-6, IL-12, and IFN-γ during DC activation. B. Significant increase in IL-2, IL-6, IL-12, and IFN-γ in the LV-SMP30 group and Protein group after co-incubation with the isolate mice PBMC, compared to the LV group or Untreated group (P<0.05). Furthermore, at one week, the levels of IL-2, IL-6, IL-12, and INF-γ in the LV-SMP30 group were significantly higher than those in the Protein group (P<0.05). The results suggest that LV-SMP30 enhances the production of IL-2, IL-6, IL-12 and IFN-γ.

The results suggest that LV-SMP30 promotes the production of IL-2, IL-6, IL-12, and IFN-γ during DC activation.

After LV-SMP30-transduced DCs were co-cultured for 72 hours with mice PBMC, the levels of IL-2, IL-6, IL-12 and IFN-γ in the supernatant were measured. As shown in Figure 4B, both IL-12 and IFN-γ in the supernatant increased more in the LV-SMP30 group and Protein group than in the LV group or Untreated group (P<0.05). However, after one week of continuous co-culture of DCs and mice PBMC, the levels of IL-2, IL-6, IL-12, and IFN-γ in the LV-SMP30 group were significantly higher than those in the Protein group (P<0.05) (Figure 4B). These results suggest that LV-SMP30-transduced DCs promote T cell differentiation and cytokine production.

LV-SMP30-transduced DCs induce CD3⁺CD8⁺ T cell activation and proliferation

Since PBMC can differentiate into T cells when stimulated with IL-2, anti-CD3, and anti-CD28, we assessed whether LV-SMP30-transduced DCs could induce the differentiation of PBMCs. We firstly isolated CD8⁺ T cells using CD8⁺ a magnetic beads separation kit, then used FCM to determine the CD3⁺CD8⁺ T cell counts. As seen in Figure 5, CD3⁺CD8⁺ T cells were the most abundant in the isolate (98.55%).

Figure 5.

The components of the isolate were assessed by flow cytometry. A. Q1-UR: CD3⁺CD8⁺ T cells; Q1-UL: CD3⁺CD8^- T cells; Q1-LL: CD3^-CD8^- T cells; Q1-LR: CD3^-CD8⁺ T cells. B. A significant difference between CD3^-CD8⁺ T cells and other cells. The result indicated that CD3⁺CD8⁺ T cells had the highest content in the isolate.

In addition, LV-SMP30-transduced DCs were more successful than other control groups at inducing CD3⁺CD8⁺ T cell proliferation based on the CCK-8 kit (Figure 6).

Figure 6.

Proliferation of CD3⁺CD8⁺ T cells was assessed by the CCK-8 kit. After the mononuclear cells were isolated, stimulatory antibodies and cytokines were added to differentiate into T cells. T cells screened with CD8⁺ immunomagnetic beads can have a purity of up to 98%. After co-culture with DCs in each group, they can be induced into CD3⁺CD8⁺ T cells. Compared to the Untreated or LV groups, the LV-SMP30 group could significantly proliferate T cells [(1.53.30±0.02)% vs. (0.18±0.02)% vs. (0.14±0.01)%, respectively, P<0.05].

CD3⁺CD8⁺ T cell-mediated cytotoxicity

As shown in Figure 7A, for H22 cells, the cytotoxicity in the LV-SMP30 group increased as the ratio of effectors/target cells increased (0:1, 10:1; 20:1; 40:1). The cytotoxicity in the LV-SMP30 group was considerably higher than that of the control groups at ratios of 10:1, 20:1 or 40:1 (P<0.05) (Figure 7B). We found no substantial cytotoxicity in each group of Hep1-6 cells, as shown in Figure 7C. The cytotoxicity of the LV-SMP30 group was not higher than that of control groups (P>0.05) (Figure 7D). These results indicate the LV-SMP30-transduced DCs enhanced the CD3⁺CD8⁺ T cell-medicated specific antitumor immune response against H22 cells.

Figure 7.

CD3⁺CD8⁺ T cell-mediated cytotoxicity was measured by flow cytometry. A. For H22 cells, the cytotoxicity in the LV-SMP30 group at the ratio of 10:1, 20:1 or 40:1. B. Statistical chart of target cell H22 specific lysis (%) in each group under different effector-target ratios. C. For Hep1-6 cells, the cytotoxicity in LV-SMP30 group at the ratio of 10:1, 20:1 or 40:1. D. Statistical chart of target cell Hep1-6 specific lysis (%) in each group under different effector-target ratios. These results indicate that LV-SMP30 transduced DCs enhances the CD3⁺CD8⁺ T cell-mediated specific cytotoxicity against H22 cells.

Construction of a liver cancer model and comparison of tumor volume

The BALB/c nude mouse model of subcutaneous xenografts was successfully constructed (Figure 8A, 8B). By analyzing the changes in tumor volume in each group, it was found that the volume in LV-SMP30 group dropped significantly with the prolongation of survival time compared to those in other control groups (P<0.05) (Figure 8C-E).

Figure 8.

Construction of BALB/c nude mouse models of subcutaneous xenografts. A. BALB/c nude mice with equal-sized (~100 mm³) single H22 tumors were treated with CD3⁺CD8⁺ T cells. B. Recording chart of tumor growth process. C. Tumor volume on day 15: PBS group, Untreated group, LV group, Protein group, LV-SMP30 group (from left to right). D. Tumors harvested from mice on day 15: PBS group, Untreated group, LV group, Protein group, LV-SMP30 group (from left to right). E. Tumor growth curve. These results demonstrated that the tumor volume in the LV-SMP30 group was smaller than that of other control groups (P<0.05).

In vivo killing mechanism of CD3⁺CD8⁺ T cells

The killing mechanism in vivo can be analyzed by measuring the secretion levels of cytokines INF-γ and IL-2 in mice serum and calculating tumor inhibition rate. Compared to the other control groups, IL-2 and IFN-γ in the LV-SMP30 group were considerably higher (P<0.05) (Figure 9A). Furthermore, the LV-SMP30 group had a greater tumor inhibitory impact than other control groups by comparing the tumor inhibition rates of each group (Figure 9B). The results suggeste that the LV-SMP30 group has a better killing effect.

Figure 9.

Killing mechanism of CD3⁺CD8⁺ T cells in BALB/c nude mouse models. A, B. Secretion levels of cytokines INF-γ and IL-2. In comparison to other control groups, IL-2 and IFN-γ in the LV-SMP30 group were increased significantly (P<0.05). C. Tumor inhibition rate. The LV-SMP30 group significantly inhibited tumor growth compared to the Untreated group and LV group. Tumor inhibition rate (%) = (average tumor weight of PBS group-average tumor weight of the experimental group)/average tumor weight of PBS group * 100%.

Discussion

Biology and immunology, particularly for tumor targets, are rapidly evolving in response to the changing time. Biological therapy represented by immunotherapy has become a popular topic in recent studies, particularly in tumor control [23]. Since anti-tumor immunity in the human body is mostly dependent on activation and proliferation, the initial T lymphocytes have a large number of aggregates in the body, on which their proliferation and killing abilities are based [24]. B cells and macrophages can interact only with memory T cells and activated cells. DCs play a particular role in the immune response because T cells are activated [25]. DCs are highly effective in antigen presentation, as a small amount of DCs activate T cells after antigen presentation [26,27]. It has been proven that DC vaccination can induce anti-tumor immunity in a variety of mouse models, so mice were used in related experiments of this study.

DC differentiation includes CD14⁺ monocyte-derived and CD34⁺ stem cells in vitro, according to the current method for generating DCs. The former is primarily detected in peripheral blood mononuclear cells (PBMCs), whereas the latter was discovered in bone marrow and cord blood [28,29]. A vast number of studies have shown that CD34⁺ stem cell-derived DCs can elicit more effective CD8⁺ T cell response than CD14⁺ monocyte-derived DCs [28,30-32]. In the present study, we derived CD34⁺ DCs from mouse bone marrow and identified LV-SMP30-transduced DCs phenotype with surface markers (CD123, CD11c, CD80 and CD86). The CD80, CD86 [33] surface molecules are all markers of DC maturation, while CD123 and CD11c are markers of DC classification in mice. The expression of CD80 and CD86 were significantly up-regulated, implying that the LV-SMP30-transfected DCs can increase the expression of mouse DC maturation.

Another essential marker of DC maturation is the secretion of cytokines, such as IL-12. High levels of IL-12 can differentiate Th0 cells into Th1 cells while promoting proliferation of Th1 cells, mediating cellular immune effects, and promoting the activation and proliferation of effector cells (NK cells, lymphokines and macrophages, etc.). They induce a variety of cytokines such as IFN-γ and IL-6 [34,35]. IL-12 and IFN-γ are both major inflammatory factors. IFN-γ can induce the expression of MHC class II molecules, and the mediation of CD3⁺CD8⁺ T cell responses can also upregulate MHC class I molecules. Our results revealed that LV-SMP30 improved the production of IL-2, IL-6, IL-12, and INF-γ. The levels of IL-2, IL-6, IL-12, and INF-γ in the LV-SMP30 group were significantly higher than those in the Protein group, especially after the DCs were cultured for continuous days. These results suggest that LV-SMP30 enhances DC maturation and secretion of Th1 cytokines, and that SMP30 is more effective at sensitizing DCs at the gene level than at the protein level.

CD3⁺CD8⁺ T cells play a crucial role in specific antitumor immunity. The co-incubation of mature DCs with T cells induces the formation and proliferation of tumor-specific CD3⁺CD8⁺ T cells. CD3⁺CD8⁺ T cells mediate the death of target cells mainly by releasing toxic particles and activating death receptor pathways. Perforin and granzymes are major cytotoxic proteins of effector cells [36,37]. When effector cells come into contact with host cells infected with pathogenic bacteria or cancerous tissues, they produce granzymes and perforin, causing host cells to die [38]. Therefore, the secretion of IL-2 and IFN-γ [39-42] cytokines reflects the activity and cytotoxicity of T cells. The ability of LV-SMP30-transduced DCs to induce the activation and proliferation of T cells was then tested. We found that LV-SMP30-transduced DCs strongly promoted the differentiation and proliferation of CD3⁺CD8⁺ T cells in mouse PBMCs. The most commonly used assay for CD3⁺CD8⁺ T cells is to detect IFN-γ, a CD3⁺CD8⁺ T cell activation marker, but other cytokines such as IL-2, IL-6, and IL-12 can also be used to differentiate activated T cells. Most importantly, the CD3⁺CD8⁺ T cell-mediated cytotoxicity was considerably higher in the LV-SMP30 group compared to those in the control groups in H22 cells but not Hep1-6 cells. These findings suggest that LV-SMP30-transduced DCs enhanced T cell activation and the specific immune response against specific mouse liver cancer H22 cells.

BALB/c nude mice lack thymus and are unable to differentiate to T cells normally. Literature reports have shown that BALB/C nude mice have lower mortality and are less expensive than SCID mice, so they are more suitable for subcutaneous liver cancer models [43,44]. Therefore, to attain the desired goal, we chose the BALB/c nude mice to construct a model of liver cancer. Besides, related literature has revealed that mice models can be constructed in a variety of ways, such as intraperitoneal injection, subcutaneous injection, and surgical transplantation. Axillary, abdominal, and other subcutaneous injection sites are available. We successfully established the BALB/c node mice subcutaneous xenograft model [45].

Next, we evaluated the changes in tumor volume and weight in each group of mice, as well as detected the serum cytokines IL-2 and IFN-γ, all of which demonstrated that the LV-SMP30 group had a stronger tumor inhibition impact than the other groups. Tumor shrinkage was inversely proportional to the rise in cytokines such as IL-2 and IFN-γ, and directly proportional to the apoptosis of liver cancer cells. The agreement with the FCM killing results in vitro showed better antitumor effects both in vitro and in vivo. Finally, we can confirm the aforesaid findings by evaluating the tumor development curve and calculating the tumor inhibition rate of each group. There are a variety of approaches to evaluate CD3⁺CD8⁺ T cells intervening in tumor growth. At present, we only measured cytokines, which is insufficient to fully comprehend the relevant mechanism. To prove this, more immunohistochemistry and immune infiltration will be studied [46]. In addition, a small animal imaging system can be used to observe the growth of cancer cells in vivo and the process of tumor reduction after intervention, which is more convenient and accurate, and saves the time and effort of killing mice at different stages, which is more ethical and animal friendly.

In conclusion, we successfully constructed and transduced mouse DCs with LV-SMP30 recombinant expressing SMP30 gene. Experimental results indicated that the LV-SMP30 group had a higher influence on DC maturation than the Untreated group and LV group, and that the LV-SMP30-transduced DCs induced CD3⁺CD8⁺ T cells to express more IL-2, IL-6, IL-12, and IFN-γ. The LV-SMP30-transduced DCs were effective at inducing special T cell cytotoxicity against mice liver cancer cells. Our results revealed that using SMP30-modified DCs could be an immunotherapy strategy for liver cancer. Moreover, we can evaluate the effect of DC-based SMP30 antigen vaccine in a mouse model to verify the previous conclusion. Finally, we will continue to evaluate this approach by constructing a mouse PDX model in a future study.

Disclosure of conflict of interest

None.