Dendritic cell vaccine as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and relates to a dendritic cell vaccine as well as a preparation method and application thereof, in particular to a dendritic cell vaccine based on enhancement of dendritic cell maturation and vaccine efficiency and application of the dendritic cell vaccine in tumor treatment. The dendritic cell disclosed by the invention is a dendritic cell with deletion of a metabotropic glutamate receptor 4 or a dendritic cell treated by forskolin, and the dendritic cell can promote the expression of co-stimulatory molecules on the surface of the dendritic cell; the proliferation of T cells is promoted; inducing the effector T cells to secrete interferon gamma and granzyme B; the killing capability of T cells on tumor cells is enhanced. The dendritic cell vaccine disclosed by the invention comprises dendritic cells loaded with antigens, wherein the antigens are liver cancer cell lysates. The dendritic cell vaccine disclosed by the invention has an anti-tumor effect and can be used for preparing anti-tumor medicines.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and relates to a dendritic cell vaccine and a preparation method and application thereof, and specifically relates to a dendritic cell vaccine based on enhancing dendritic cell maturation and vaccine efficacy and an application thereof in tumor treatment. Background Art

[0002] Tumor immunotherapy is a treatment method that fights cancer by enhancing or restoring the patient's immune system. Unlike traditional surgery, radiotherapy and chemotherapy, immunotherapy activates the patient's own immune system to identify and attack tumor cells, so it is highly specific and long-lasting. Tumor immunotherapy methods include immune checkpoint inhibitors, cytokine therapy, adoptive cell therapy, and vaccine immunotherapy.

[0003] Tumor immunotherapy is a treatment method that mobilizes the patient's own immune system to identify and eliminate cancer cells. In recent years, dendritic cells (DCs) have become a research hotspot in cancer immunotherapy due to their core role in antigen presentation and T cell activation. DCs are the most effective antigen-presenting cells (APCs) in mammals, and are mainly responsible for capturing, processing and presenting antigens, initiating and regulating T cell-mediated immune responses. Dendritic cells originate from the bone marrow, exist in an immature form in the blood and peripheral tissues, and have a strong ability to capture antigens. After dendritic cells capture antigens, they mature and migrate to the lymph nodes after specific signal stimulation, where they come into contact with T cells, activate T cells and initiate adaptive immune responses.

[0004] The function of dendritic cells is closely related to their maturity state. Immature dendritic cells are mainly responsible for capturing and processing exogenous antigens, while mature dendritic cells have a strong T cell activation ability and can present antigen information to T cells by expressing co-stimulatory molecules (such as CD80, CD86) and major histocompatibility complex (MHC) molecules, thereby inducing T cell proliferation and differentiation and initiating specific immune responses. Dendritic cells play a key role in anti-tumor immune responses by recognizing and presenting tumor antigens, activating cytotoxic T cells (CTLs), and effectively killing tumor cells.

[0005] By culturing the patient's dendritic cells in vitro, loading them with specific tumor antigens, and then injecting them back into the patient, the dendritic cells can perform the function of antigen presentation in vivo and activate the patient's T cells to attack tumor cells. Compared with traditional vaccines, dendritic cell vaccines have stronger personalization characteristics and can be customized according to the specific tumor antigens of each patient. However, the current dendritic cell vaccines still have some problems in clinical applications, such as the maturity of DCs and the immune activation effect are not strong enough, resulting in limited immune effects of the vaccine. Therefore, how to optimize the function of dendritic cells to improve their application effect in tumor immunotherapy has become a research focus. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention first provides a dendritic cell, wherein the dendritic cell is a dendritic cell lacking metabotropic glutamate receptor 4 (mGluR4) (encoding gene Grm4) or a dendritic cell treated with Forskolin;

[0007] Furthermore, the dendritic cells are mGluR4-deficient dendritic cells induced by GM-CSF or dendritic cells treated with Forskolin induced by GM-CSF.

[0008] The dendritic cells are highly mature and have strong T cell activation ability. They can present antigen information to T cells by expressing co-stimulatory molecules (such as CD80, CD86) and major histocompatibility complex (MHC) molecules, thereby inducing T cell proliferation and differentiation and initiating specific immune responses.

[0009] The GM-CSF-induced forskolin-treated dendritic cells are prepared by the following method:

[0010] Forskolin was added to the GM-CSF-differentiated dendritic cell culture system and cultured for 24-48 hours;

[0011] Wherein, the mass volume concentration of GM-CSF is 15-25 ng / ml; the molar concentration of forskolin is 15-25 μM;

[0012] Furthermore, the ratio of the mass volume concentration of GM-CSF to the molar concentration of forskolin is 1:1 (ng / ml:μM).

[0013] The second object of the present invention is to provide a dendritic cell vaccine, wherein the dendritic cell vaccine comprises dendritic cells loaded with antigens, and the antigens are liver cancer cell lysates.

[0014] The antigen is prepared by the following method:

[0015] Hepa1-6BL liver cancer cells were obtained by trypsin digestion, and the cell pellets were obtained after centrifugation. The cells were then resuspended in culture medium and the tumor cells were repeatedly frozen and thawed five times.

[0016] The dendritic cells are dendritic cells with mGluR4 deletion induced by granulocyte-macrophage colony stimulating factor or dendritic cells treated with forskolin induced by granulocyte-macrophage colony stimulating factor.

[0017] The mass ratio of the dendritic cells to the antigen is 1:1-1:2.

[0018] The third object of the present invention is to provide a method for preparing a dendritic cell vaccine, comprising the following steps:

[0019] The dendritic cell vaccine is obtained by using GM-CSF-induced mGluR4-deficient dendritic cells or GM-CSF-induced forskolin-treated dendritic cells and antigen loading.

[0020] The mass volume concentration of GM-CSF is 15-25 ng / ml; the molar concentration of forskolin is 15-25 μM.

[0021] The antigen is a liver cancer cell lysate, and the mass ratio of the dendritic cells to the antigen is 1:1-1:2.

[0022] The fourth object of the present invention is to provide the use of the dendritic cell vaccine in the preparation of anti-tumor drugs.

[0023] A fifth object of the present invention is to provide a use of mGluR4-deficient dendritic cells or forskolin-treated dendritic cells in promoting the maturation and function of dendritic cells.

[0024] The dendritic cells of the present invention can promote the expression of co-stimulatory molecules on the surface of dendritic cells; promote the proliferation of T cells; induce effector T cells to secrete interferon gamma (IFNγ) and granzyme B (Granzyme B); and enhance the killing ability of T cells on tumor cells.

[0025] The sixth object of the present invention is to provide an immune adjuvant, wherein the immune adjuvant is forskolin.

[0026] Furthermore, the present invention provides the use of the forskolin as an immune adjuvant in the preparation of dendritic cell vaccines.

[0027] The mGluR4-deficient dendritic cells of the present invention can promote the expression of dendritic cell co-stimulatory molecules CD80, CD86 and MHCII, and further enhance the antigen-specific OT1 CD8 + T cell proliferation, secretion of IFNγ and Granzyme B, and killing of tumor cells.

[0028] The mGluR4 deletion strategy provided by the present invention can enhance the maturation and function of dendritic cells and thus promote the efficacy of dendritic vaccines. Specifically, mGluR4-deficient dendritic cells can be adopted into mice bearing orthotopic liver cancer, which can significantly inhibit the growth of tumors in the liver and spontaneous lung metastasis in mice.

[0029] It can be seen that mGluR4 can be used as a tumor marker to diagnose tumors and evaluate the prognosis of tumors.

[0030] mGluR4 can be used as a drug target in the development, design, research and screening of anti-tumor drugs.

[0031] mGluR4 inhibitors can enhance the maturation and function of dendritic cells, promote the expression of dendritic cell co-stimulatory molecules CD80, CD86 and MHCII, and further enhance the antigen-specific OT1 CD8 + T cell proliferation, secretion of IFNγ and Granzyme B, and killing of tumor cells.

[0032] The dendritic cell vaccine of the present invention can be used to prepare anti-tumor drugs.

[0033] The specific steps include: constructing a mouse tumor model, administering adoptive DC vaccine cells, and monitoring the tumor growth in mice.

[0034] The mouse tumor model is a mouse in situ liver cancer model, which adopts a high-pressure tail vein water injection method.

[0035] When adopting dendritic cell vaccine, the number of adoptive cells is 3x10 6 cells per mouse.

[0036] Forskolin is a natural compound derived from the plant Coleus forskohlii, and its main mechanism of action is to promote the production of intracellular cyclic adenosine monophosphate (cAMP) by activating adenylate cyclase (AC). cAMP is an important second messenger in cells and plays a key role in the signal transduction process of various cells. In particular, in immune cells, the activation of the cAMP signaling pathway is of great significance for the functional regulation of cells. The present invention uses forskolin as an immune adjuvant to enhance the maturation and function of dendritic cells.

[0037] Forskolin can be used as an AC agonist and can modulate the functional characteristics of dendritic cells, showing good application prospects in optimizing the anti-tumor immune effects of dendritic cell vaccines.

[0038] The present invention enhances the maturation and function of dendritic cells by adding immune adjuvant Forskolin or by using the strategy of mGluR4 deletion.

[0039] Studies have shown that forskolin treatment can promote dendritic cells to express more mature markers, such as CD80, CD86, MHC class I and class II molecules, and increase their secretion of pro-inflammatory cytokines (such as IL-12, IFN-γ). These molecules and cytokines play a crucial role in the activation of T cells, allowing DCs to more effectively initiate specific immune responses against tumor antigens.

[0040] mGluR4-deficient DCs can promote the expression of DC co-stimulatory molecules CD80, CD86 and MHCII, further enhancing antigen-specific OT1 CD8 + T cell proliferation, secretion of IFNγ and Granzyme B, and killing of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a schematic diagram of the results of detecting the regulation of the expression of dendritic cell co-stimulatory molecules CD80, CD86 and MHC II by mGluR4 deficiency by flow cytometry in Example 1 of the present invention.

[0042] a: Representative images of flow cytometry detection of the regulation of mGluR4 deficiency on the expression of dendritic cell co-stimulatory molecules CD80, CD86 and MHC II.

[0043] b: Statistical analysis of the effects of mGluR4 deficiency on the expression of dendritic cell co-stimulatory molecules CD80, CD86 and MHC II detected by flow cytometry.

[0044] Figure 2 The OT1CD8 mediated by mGluR4-deficient dendritic cells detected by flow cytometry in Example 2 of the present invention + Schematic diagram of the effects on T cell proliferation.

[0045] a:OT-1CD8 + Representative peak graphs of T cell proliferation;

[0046] b: Statistical analysis of OT-1CD8 T cell subsets;

[0047] Clusters 1, 2, and 3 represent CD8 +The number of T cell divisions, 0 means no proliferation of CD8 + T cells;

[0048] c: CD8 after culture + The number of T cells.

[0049] Figure 3 In Example 3 of the present invention, flow cytometry was used to detect the mGluR4-deficient dendritic cells mediated the OT1CD8 + Schematic diagram of the results of T cell antigen presentation ability.

[0050] a: Flow cytometry showing co-cultured CD8 + Representative graphs of granzyme B (GzmB) and IFNγ expression levels in T cells;

[0051] b: CD8 expressing GzmB and IFNγ + Statistical analysis of the proportion of T cells to total CD8 T cells;

[0052] c: Detect CD8 using ELISA kit + IFNγ levels in the cell culture supernatant after co-culture of T cells and dendritic cells.

[0053] Figure 4 The OT1CD8 mediated by mGluR4-deficient dendritic cells detected by flow cytometry in Example 4 of the present invention + Schematic diagram of the results of T cell regulation of liver cancer cell killing ability.

[0054] a: Flow cytometry showed control and Grm4 - / - After dendritic cells and CD8 T cells were co-cultured, CD8 + Representative images of T cells killing Hepa1-6BL cells (analysis of the level of Annexin V expressed by tumor cells reflects the apoptosis of tumor cells and evaluates the CD8 + T cells’ ability to kill tumor cells);

[0055] b: Apoptosis of Hepa1-6BL reflects the apoptosis of WT and Grm4 - / - CD8 dendritic cells after co-culture + Statistical graph of T cell killing ability.

[0056] Figure 5 This is a schematic diagram of the results of Example 5 of the present invention, in which adoptive transfer of mGluR4-deficient dendritic cells inhibited the growth of orthotopic hepatocellular carcinoma and spontaneous lung metastasis in tumor-bearing mice.

[0057] a: Representative images of the liver of tumor-bearing mice;

[0058] b: Statistical analysis of liver tumors in tumor-bearing mice, the total number of original liver cancer nodules, and the number of liver tumor nodules of different sizes.

[0059] Figure 6 For example 5 of the present invention, different Schematic diagram of the results showing that the levels of ALT and AST in the serum of tumor-bearing mice were used to indicate the liver damage of mice. The adoptive transfer of mGluR4-deficient dendritic cells reduced the liver damage of tumor-bearing mice.

[0060] Figure 7 This is a schematic diagram of the results of detecting the regulation of the expression of dendritic cell co-stimulatory molecules CD80, CD86 and MHC II by AC agonist forskolin treatment by flow cytometry in Example 6 of the present invention.

[0061] a: Representative images of the regulation of the expression of co-stimulatory molecules CD80, CD86 and MHC II of dendritic cells by Forskolin treatment detected by flow cytometry;

[0062] b: Statistical analysis of the effect of Forskolin treatment on the proportion of dendritic cells expressing co-stimulatory molecules CD80, CD86 and MHC II;

[0063] c: Statistical analysis of the effect of Forskolin treatment on the expression intensity of CD80, CD86 and MHC II in dendritic cells (mean fluorescence intensity of cells analyzed by flow cytometry).

[0064] Figure 8 The OT1 CD8 mediated by dendritic cells treated with AC agonist forskolin was detected by flow cytometry in Example 7 of the present invention. + Schematic diagram of the effects on T cell proliferation.

[0065] a: Forskolin treatment of dendritic cells and CD8 + T cell co-culture and flow cytometry detection of CD8 + Representative peak graphs of T cell proliferation;

[0066] b:CD8 + Statistical analysis of T cell subsets;

[0067] Clusters 1, 2, and 3 represent CD8 + The number of T cell divisions, 0 means no proliferation of CD8 + T cells;

[0068] c: Total number of CD8 T cells after co-culture.

[0069] Fig. 9The AC agonist forskolin-treated dendritic cells mediated the OT1 CD8 + Schematic diagram of the results of T cell antigen presentation ability.

[0070] a: Flow cytometry showing co-cultured CD8 + Representative graphs of granzyme B (GzmB) and IFNγ expression levels in T cells;

[0071] b: CD8 expressing GzmB and IFNγ + The proportion of CD8 T cells in total CD8 T cells and the CD8 + Statistical analysis of mean fluorescence intensity in T cells;

[0072] c: The level of IFNγ in the cell culture supernatant after co-culture of CD8 T cells and dendritic cells was detected using an ELISA kit.

[0073] Fig.10 The OT1 CD8 mediated by dendritic cells treated with AC agonist forskolin was detected by flow cytometry in Example 9 of the present invention. + Schematic diagram of the results of T cell regulation of liver cancer cell killing ability.

[0074] a: Flow cytometry showing control and Forskolin-treated dendritic cells and CD8 + After co-culture of T cells, CD8 + Representative images of T cells killing Hepa1-6BL cells (analysis of the level of Annexin V expressed by tumor cells reflects the apoptosis of tumor cells and evaluates the CD8 + T cells’ ability to kill tumor cells);

[0075] b: Apoptosis of Hepa1-6BL reflects the CD8 + Statistical graph of T cell killing ability.

[0076] Fig.11 This is a schematic diagram of the results of Example 10 of the present invention, in which dendritic cells treated with the AC agonist forskolin inhibited the growth of orthotopic hepatocellular carcinoma and spontaneous lung metastasis in tumor-bearing mice.

[0077] a: Representative images of the liver of tumor-bearing mice;

[0078] b: Statistical analysis of liver tumors in tumor-bearing mice, the total number of original liver cancer nodules, and the number of liver tumor nodules of different sizes;

[0079] c: Representative images and statistical analysis of spontaneous lung metastasis from HCC in situ.

[0080] Fig.12 For example 11 of the present invention, different Schematic diagram of the results using ALT and AST levels in the serum of tumor-bearing mice to indicate the liver damage of mice. Adoptive transfer of dendritic cells treated with the AC agonist forskolin reduced liver damage in tumor-bearing mice.

[0081] As shown in the above figures, *: p<0.05 **: p<0.01 ***: p<0.005 ****: p<0.001. DETAILED DESCRIPTION

[0082] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The test methods described in the following embodiments are all conventional methods unless otherwise specified; the consumables involved can be obtained through commercial channels unless otherwise specified.

[0083] The materials and sources used in the following examples are as follows: RPMI 1640 medium (VICMED); FBS (CLARK); Glutamine (1-4 mmol / L), 2-ME (50 μM), NEAA (10 mM), HEPES (1 M / ml); GM-CSF (CHAMOT); Recombinant human IL-2 protein (Beijing T&L Biotechnology); LPS (Sigma); Anti-CD11c, Anti-CD11b, Anti-CD80, Anti-CD86, Anti-MHCII, Anti-CD4, Anti-CD8, Anti-TCRβ, Anti-IFNγ, Anti-Granzyme B, 7AAD (Clone N / A); Zombie Aqua TM(Clone N / A)(Elabscience,BioLegend); 123count eBeads ebioscience(BioLegend); Anti-CD3(Elabscience); Mouse IFN-γ(Interferon Gamma)ELISAKit(Elabscience); PMA(MCE); Ionomycin(MCE); Brefeldin A(MCE); Annexin V Binding Buffer(BioLegend); FITCAnnexin V Apoptosis Detection Kit(BioLegend); OVA peptide(257-264)(SangonBiotech); CellTrace TM Violet (Invitrogen); Carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Beyotime); Alanine Aminotransferase (ALT) Activity Assay Kit (Solarbio); Aspartate Aminotransferase (AST) Activity Assay Kit (Solarbio); Forskolin (Aladdin); RPMI 1640 medium (VICMED).

[0084] Preparation of mGluR4-deficient (Grm4 KO) dendritic cells:

[0085] The px459-Cas9-puro vector was digested with BbsI to make it linear. CRISPR primers targeting mouse Grm4 exon 2 were designed (sgGrm4-1: (5'-CAGCCGAGCCGTTGCGACGTTGG-3');

[0086] sgGrm4-2: (5'-ATCGACCGGTGTACGCCATGGG-3').

[0087] The forward and reverse oligonucleotides of each primer were annealed using a standard annealing protocol, cloned into the px459-Cas9-puro vector by T4 ligation, and amplified and sequenced to obtain the target vector plasmid. The DNA concentration was determined using a nanodrop spectrophotometer. Dendritic cells were plated at an appropriate density, and a DNA-transfection reagent complex solution was prepared according to the instructions. The dendritic cells were transiently transfected with the target gene vector plasmid using Lipo2000 transfection reagent to produce CRISPR-Cas9-mediated Grm4 gene knockout dendritic cells. Cells were treated with puromycin for screening, and the ablation of the target gene was confirmed by sequencing. Mouse Grm4 KO dendritic cells were obtained. Its gene sequence is shown in SEQ ID No.1.

[0088] Human Grm4 KO dendritic cells were obtained in the same manner, and the gene sequence thereof is shown in SEQ ID No.2.

[0089] Example 1 Effect of mGluR4 deficiency on the expression of co-stimulatory molecules in mouse DC

[0090] 1. Obtaining mouse bone marrow-derived dendritic cells (BMDC)

[0091] 1.1 Take C57BL / 6J WT and Grm4 - / - Adult mice were killed and then soaked in 75% alcohol for 2-3 minutes for disinfection. An incision was made in the abdomen, and the muscles on the bones were separated with scissors. The tibia and femur of the mouse were taken and then soaked in PBS buffer, and then washed with sterile PBS several times and the excess tissue was removed (soaked and washed 5-6 times). The subsequent steps were completed in a sterile operating table.

[0092] 1.2 Use sterile tweezers to move the tibia and femur into another new culture dish containing PBS, then use a 20ml syringe to draw up sterile PBS, clamp one end of the tibia or femur with tweezers, cut off both ends of the bone with scissors, insert a 1ml syringe needle into the bone marrow cavity from both ends of the bone, and use a 20ml syringe filled with PBS to repeatedly flush the bone marrow up and down alternately. Pass the flushed bone marrow cells through a 70μm cell sieve while grinding, collect them into a 50mL sterile centrifuge tube, and centrifuge at 2000rpm for 4 minutes at 4℃.

[0093] 1.3 Add 1 ml of red blood cell lysis buffer (ACK) to the cell pellet, mix thoroughly to resuspend the cells, lyse at room temperature for about 90 seconds, add 10 ml of pre-cooled PBS for washing, and centrifuge at 4°C, 2000 rpm for 4 minutes.

[0094] 1.4 Resuspend the cells in RPMI 1640 medium, add β-mercaptoethanol and recombinant mouse GM-CSF (20 ng / ml) to the RPMI 1640 medium, and adjust the cell density to 2-3×10 6 / well, plated in untreated 6-well cell culture plates and incubated at 37°C, 5% CO 2 Cultivated under conditions.

[0095] 1.5 Observe the cell status. The cells are in a semi-adherent state in many clusters. Gently shake the culture plate and add culture medium every two days. On the fourth day, gently shake the culture plate, completely aspirate the culture supernatant, and immediately add 2 ml of fresh RPMI 1640 complete culture medium. Centrifuge the aspirated supernatant at 2000 rpm for 4 minutes, discard half of the supernatant and mix well, then add β-mercaptoethanol and recombinant mouse GM-CSF (20 ng / ml).

[0096] 1.6 Start laying out the plates on the 6th day.

[0097] 2. Flow cytometry

[0098] Expression of co-stimulatory molecules in BMDCs

[0099] 2.1 Gently pipette and collect the suspended and semi-adherent BMDC cells on day 6 into a sterile 50 ml centrifuge tube. Centrifuge at 2000 rpm for 4 min at 4°C, discard the supernatant, and add fresh BMDC culture medium to resuspend the BMDC cells to a concentration of 5 × 10 5 / ml. According to 1×10 5 / 200ul / well was plated into an untreated 48-well plate.

[0100] 2.2 Expression of WT and Grm4 - / - BMDCs were divided into two groups and plated with or without tumor cell conditioned medium (TCM) for 24 h.

[0101] 2.3 After the treatment, the cells were collected in a 96-well U-shaped plate, centrifuged at 4°C, 2000 rpm for 4 min, and the supernatant was discarded. The cells were washed once with PBS, centrifuged at 4°C, 2000 rpm for 4 min, and the supernatant was discarded.

[0102] 2.4 Prepare a mixed solution containing anti-MHCII, anti-CD80, anti-CD86 and zombie aqua (ZA, Live / dead) flow cytometry antibodies to perform flow cytometry surface staining on the cells.

[0103] 2.5 Flow cytometry was used to detect the expression levels of BMDC co-stimulatory molecules CD80, CD86 and MHCII. -The cells were living cells, and their expression levels were reflected by detecting the positive cell percentage and mean fluorescence intensity of CD80, CD86 and MHCII in BMDC.

[0104] The experimental results are as follows Figure 1 As shown in a and b, flow cytometry results showed that mGluR4 deficiency could upregulate the expression of co-stimulatory molecules CD80, CD86 and MHCII on the surface of BMDC cells.

[0105] Example 2 mGluR4-deficient DCs versus CD8 + Effects on T cell proliferation

[0106] WT or Grm4 - / - BMDCs were cultured in fresh medium (FM) or Hepa1-6BL conditioned medium (TCM) at a ratio of 1:1 for 24 h, then pulsed with 1 ng / ml of OVA peptide (SIINFEKL) for 2 h, and then co-cultured with CTV-labeled OT-1 CD8+ T cells at a ratio of 1:10. In the co-culture system, T cell proliferation, cytokine production, and cytotoxicity against tumor cells were measured after 2 days.

[0107] The specific steps are as follows:

[0108] 1. Obtaining and processing BMDC cells

[0109] 1.1 Bone marrow cells were cultured in a medium containing the cytokine GM-CSF for 6 days to obtain BMDCs with very weak immunogenicity.

[0110] 1.2 Gently pipette and collect the suspended and semi-adherent BMDC cells on day 6 into a sterile 50 ml centrifuge tube, centrifuge at 2000 rpm for 4 min at 4°C, discard the supernatant, add fresh BMDC culture medium and resuspend the BMDC cells to a concentration of 1×10 5 / ml. According to 1×10 4 / 100ul / well was plated into a 96-well plate.

[0111] 1.3 Transfection of WT and Grm4 - / - BMDCs were divided into 2 groups and plated with or without TCM for 24 h.

[0112] 2. Flow cytometry to obtain OT1 CD8 + T cells

[0113] 2.1 Take C57BL / 6J OT1 wild-type adult mice, kill them and soak them in 75% alcohol for 2-3 minutes for disinfection. Make an incision in the abdomen, separate the spleen with scissors, soak it in PBS buffer, and then soak and wash it with sterile PBS several times.

[0114] 2.2 Grind the spleen with the handle of a 1 ml syringe and rinse the spleen repeatedly with a 20 ml syringe filled with PBS. Pass the spleen cells through a 70 μm cell sieve while rinsing and grinding, collect them into a 50 ml sterile centrifuge tube, and centrifuge at 2000 rpm for 4 min at 4°C.

[0115] 2.3 Add 1 ml of red blood cell lysis buffer (ACK) to the cell pellet, mix thoroughly to resuspend the cells, lyse at room temperature for about 90 seconds, add 10 ml of pre-cooled PBS for washing, and centrifuge at 4°C, 2000 rpm for 4 minutes.

[0116] 2.4 Prepare a mixed solution containing flow cytometry antibodies such as anti-TCRβ and anti-CD8a, and perform flow cytometry surface staining on the cells.

[0117] 2.5 After staining, add 7AAD (Live / dead) antibody and dilute it with FACS (PBS solution containing 2% serum), and sort OT1 CD8 by flow cytometry. + T cells.

[0118] 2.6 The CD8 + T cells were centrifuged at 2000 rpm for 4 min at 4°C and added to CellTrace TM Violet for CD8 + T cell labeling and staining for 15 min; then 10 ml of PBS was added to terminate the staining, and the cells were centrifuged at 2000 rpm for 4 min at 4°C.

[0119] 3.BMDC and CD8 + T cell co-culture

[0120] 3.1 BMDC culture supernatant was discarded and 1 ng / ml OVA peptide (257-264) was added to stimulate for 2 h, then the stimulation supernatant was discarded and washed once with PBS, and the obtained CD8 + T cells.

[0121] 3.2 Fresh RPMI 1640 complete medium to obtain CD8 + T cells were resuspended to a concentration of 5 × 10 5 / ml, supplemented with 100ng / ml human recombinant IL2. 5 / 200ul / well was re-plated into a 96-well flat-bottom plate containing BMDC cells and placed in 37°C, 5% CO 2 The cells were cultured in an incubator for 2 days.

[0122] 3.3 After proliferation, add 7AAD antibody and beads, and detect CD8 by flow cytometry +T cell proliferation peak and cell number. Figure 2 As shown in Figures ac, mGluR4 deficiency enhances dendritic cell responses to CD8 + Example 3 Effect of mGluR4-deficient DC on CD8 + Effects of IFNγ and Granzyme B on T cell effector molecules 1.CD8 + T cell stimulation

[0123] 1.1 Combine the above BMDC with CD8 + After T cells proliferated, PMA, Ionomycin and GolgiPlug stimulators were added for 4 hours.

[0124] 2. CD8 + T cell intracellular staining and flow cytometry

[0125] 2.1 After stimulation, CD8 + T cells were collected in a 96-well U-plate, centrifuged at 4°C, 2000 rpm for 4 min, and the supernatant was discarded. The cells were washed once with PBS, centrifuged at 4°C, 2000 rpm for 4 min, and the supernatant was discarded.

[0126] 2.2 Prepare a mixture containing zombie aqua (ZA, Live / dead) flow cytometry antibody and perform flow cytometry surface staining on cells.

[0127] 2.3 Centrifuge the cells at 4°C, 2000 rpm for 4 min, discard the supernatant, add fixative and fix on ice for 30 min.

[0128] 2.4 Use the punching solution to prepare a flow cytometry antibody mixture containing anti-IFNγ and anti-Granzyme B for intracellular staining of cells.

[0129] 2.5 Detection of CD8 by flow cytometry + Expression levels of IFNγ and anti-Granzyme B in T cells.

[0130] The experimental results are as follows Figure 3 As shown in a and b, flow cytometry analysis showed that mGluR4 deficiency could enhance CD8 + Expression of IFNγ and anti-Granzyme B in T cells.

[0131] 3.BMDC and CD8 + IFNγ secretion in T cell co-culture system

[0132] Charge BMDC and CD8 + The cell culture supernatant after 48 h of T cell co-culture was used to detect the secretion of IFNγ in the co-culture system using a mouse IFNγ ELISA detection kit.

[0133] The results are as follows Figure 3 As shown in c, mGluR4 deficiency can enhance CD8 + Secretion of IFNγ by T cells.

[0134] Example 4 mGluR4-deficient DCs and CD8 + Effect of T cell killing ability

[0135] 1. Treatment of Tumor Cells

[0136] 1.1 When the Hepa1-6BL cell density reaches 80%-90%, cell passage is performed in a clean bench as follows: discard the cell culture supernatant, add 3 ml of sterile PBS buffer to wash the cells, discard the PBS, add 0.5 ml of 0.25% EDTA trypsin digestion solution to completely cover the bottom of the cell culture dish, and place it in a 37°C, 5% CO 2 Incubator with 4% paraformaldehyde and digest for 1 min. Observe the cell morphology under an inverted microscope. When some cells fall off or the cell morphology changes, add 2 ml of complete culture medium to the culture dish and blow the cells at the bottom of the culture dish to make them fall off completely. Transfer the cells to a centrifuge tube and centrifuge at 350 g for 4 min at 4°C. Discard the supernatant.

[0137] 1.2 The Hepa1-6BL cells obtained by centrifugation were labeled with CFSE and stained for 15 minutes; then 10 ml of PBS was added to terminate the staining and centrifuged at 2000 rpm for 4 minutes at 4°C.

[0138] 1.3 Add fresh RPMI 1640 complete medium and resuspend the CFSE-labeled Hepa1-6BL cells to a concentration of 2×10 5 / ml. According to 2×10 4 / 100ul / well re-plate into 96-well flat-bottom plates and place in 37°C, 5% CO 2 cultured in an incubator.

[0139] 2. Effect of mGluR4 deficiency on BMDC-mediated CD8 + The killing ability of T cells on Hepa1-6BL hepatocellular carcinoma cells

[0140] 2.1 BMDC and CD8 + CD8 T cells proliferated in co-culture system for 48 h + T cells were collected in a 1.5 ml centrifuge tube, centrifuged at 2000 rpm for 4 min at 4°C, and the supernatant was discarded.

[0141] 2.2 Add fresh RPMI 1640 complete medium to CD8 +T cells were resuspended to a concentration of 5 × 10 5 / ml. According to 1×10 5 / 200ul / well was added back into a 96-well flat-bottom plate containing Hepa1-6BL cells and placed in 37°C, 5% CO 2 Incubate in an incubator for 18 h.

[0142] 2.3 After the co-culture, the cells were digested with trypsin, collected in a 96-well U-shaped plate, centrifuged at 2000 rpm for 4 min at 4°C, and the supernatant was discarded.

[0143] 2.4 Prepare Annexin V flow cytometry antibody solution with Annexin V buffer and perform flow cytometry staining on the cells for 15 minutes.

[0144] 2.5 Detection of CD8 by flow cytometry + The killing ability of T cells on Hepa1-6BL cells.

[0145] The experimental results are as follows Figure 4 As shown in a and b, flow cytometry analysis showed that mGluR4 deficiency could enhance BMDC-mediated CD8 + T cells kill Hepa1-6BL liver cancer cells.

[0146] Example 5 DCs lacking mGluR4 inhibit the growth of orthotopic hepatocellular carcinoma in mice

[0147] Adoptive transfer of mGluR4-deficient dendritic cells inhibited the growth and spontaneous lung metastasis of orthotopic hepatocellular carcinoma in tumor-bearing mice. 5 Hepa1-6BL tumor cells were hydrodynamically injected into C57BL / 6WT mice via the tail vein, with 6-9 mice in each group. On days 3, 9, and 15 relative to tumor cell inoculation, WT or Grm4 loaded with Hepa1-6BL tumor cell lysate were injected - / - BMDC (3×10 6 ). The mice were sacrificed on day 21, and the livers were collected for tumor burden quantification.

[0148] The specific steps are as follows:

[0149] 1. Preparation of Tumor Lysate

[0150] 1.1 When the Hepa1-6BL cell density reaches 80%-90%, collect the Hepa1-6BL cells, resuspend them in fresh RPMI1640 complete medium and freeze-thaw them 6 times (-80°C for 30 min, thaw in a 37°C water bath).

[0151] 2. Preparation of DC Vaccine

[0152] 2.1 Gently pipette and collect the suspended and semi-adherent BMDC cells on day 6 into a sterile 50 ml centrifuge tube. Centrifuge at 2000 rpm for 4 min at 4°C, discard the supernatant, and add fresh BMDC culture medium to resuspend the BMDC cells to a concentration of 2×10 6 / ml. According to 4×10 6 / 2ml / well was plated into untreated 6-well plates.

[0153] 2.2 Expression of WT and Grm4 - / - BMDCs were divided into 2 groups and plated with or without TCM for 24 h.

[0154] 2.3 The tumor cell lysate obtained above was added to BMDCs at a ratio of 1:1 tumor cell:BMDC (approximately one tumor cell lysate per BMDC). After pulsing with tumor lysate for 16 h in growth medium containing BMDCs, the cells were harvested, washed thoroughly with sterile PBS, and resuspended in PBS.

[0155] 3. Construction of Mouse Tumor Model

[0156] 3.1 Construction of liver cancer in situ in mice: When the density of Hepa1-6BL cells reached 80%-90%, the cells were passaged in a clean bench as follows: discard the cell culture supernatant, add 3 ml of sterile PBS buffer to wash the cells, discard the PBS, add 0.5 ml of 0.25% EDTA trypsin digestion solution to completely cover the bottom of the cell culture dish, and place it in a 37°C, 5% CO 2 Incubator with 4% paraformaldehyde and digest for 1 min. Observe the cell morphology under an inverted microscope. When some cells fall off or the cell morphology changes, add 2 ml of complete culture medium to the culture dish and blow the cells at the bottom of the culture dish to make them fall off completely. Transfer the cells to a centrifuge tube and centrifuge at 350 g for 4 min at 4°C. Discard the supernatant.

[0157] 3.2 Add the Hepa1-6BL cells obtained by centrifugation into fresh RPMI 1640 basal medium and resuspend the cells to a concentration of 3.3×10 5 / ml.

[0158] 3.3 According to 5×10 5 Hepa1-6BL / 1.5 ml / mouse was injected into C57BL / 6J WT and Grm4 cells via tail vein injection. - / - Adult mice were divided into WT BMDC control group and Grm4 - / - BMDC-treated group.

[0159] 4. Injection of DC Vaccine into Tumor-bearing Mice

[0160] 4.1 After the pulse is over, gently pipette to collect the suspended and semi-adherent BMDC cells on day 6 into a sterile 50 ml centrifuge tube, centrifuge at 4°C, 2000 rpm for 4 min, discard the supernatant, add fresh RPMI 1640 basal medium and resuspend the BMDC cells to a concentration of 4×10 7 / ml.

[0161] 4.2 On days 3, 9, and 15 after tumor injection, 200 μl of the 2×10 6 Antigen-loaded BMDCs.

[0162] 4.3 At the end of the 21st day of the mouse tumor-bearing experiment, the mouse body weight, liver tumor weight, liver tumor nodules, serum and spontaneous lung metastasis were counted.

[0163] 5. Detection of serum AST and ALT in tumor-bearing mice

[0164] 5.1 At the end of the 21st day of the experiment, blood samples were collected from mice with orthotopic liver tumors. The samples were stored at room temperature and centrifuged at 3000 rpm for 10 min to obtain mouse serum.

[0165] 5.2 The levels of alanine aminotransferase (ALT), also known as alanine aminotransferase (GPT) activity assay kit and aspartate aminotransferase (AST), also known as aspartate aminotransferase (GOT) assay kit were used to measure the levels of alanine aminotransferase and aspartate aminotransferase in serum aliquots. The above biochemical parameters were tested according to the manufacturer's instructions.

[0166] The results showed that: a high-pressure liver orthotopic tumor model was established, and DC vaccine was adopted by tail vein. Figure 5 As shown in a and b, the growth of orthotopic hepatocellular carcinoma tumors in mice adoptively transplanted with mGluR4-deficient BMDC cells was significantly inhibited.

[0167] The mouse serum was further analyzed and the results were as follows Figure 6 As shown, the levels of AST and ALT in the serum of mice adoptively transplanted with mGluR4-deficient BMDC cells were significantly reduced, indicating that the adoptive transplantation of mGluR4-deficient BMDC cells reduced the degree of liver damage in mice.

[0168] Glutamatergic signaling is key to normal nervous system function and regulates innate immunity through receptors expressed in immune cells. Metabotropic glutamate receptors (mGluRs) belong to the G protein-coupled receptor family C, which consists of eight members, mGluR1 to mGluR8, and is divided into three groups. mGluRs are activated by glutamate and couple to intracellular G proteins to mediate intracellular second messenger signals, participating in the regulation of neuronal excitation and synaptic transmission. mGluR4 (encoded by Grm4) belongs to the mGluRs family III, couples to Gi and Go proteins, is a presynaptic glutamate receptor, expressed in the basal ganglia circuits that control movement, and participates in the neurotransmission of presynaptic excitatory glutamate signals. The role of mGlur4-mediated glutamatergic signaling in regulating immune responses has been reported. Mice lacking mGluR4 have altered characteristics of cytokine production in DCs and are highly sensitive to experimental autoimmune encephalomyelitis (EAE, a mouse model of multiple sclerosis).

[0169] The results of Examples 1-5 show that the DC vaccine with mGluR4 deletion can significantly inhibit the growth of orthotopic liver cancer in C57BL / 6J wild-type mice. The vaccine is simple to prepare, has clear ingredients, can effectively and quickly promote DC cell maturation and antigen presentation, and induce the proliferation, activation and function of cytotoxic T lymphocytes (CTLs).

[0170] Example 6 Effect of Forskolin on the Expression of Co-stimulatory Molecules in Mouse DC

[0171] 1. Obtaining mouse bone marrow-derived dendritic cells (BMDC)

[0172] 1.1 Take C57BL / 6J wild-type adult mice, kill them and soak them in 75% alcohol for 2-3 minutes. Make an incision in the abdomen, separate the muscles on the bones with scissors, take the mouse tibia and femur, soak them in PBS buffer, and then soak and wash them with sterile PBS several times and remove excess tissue (soak and wash 5-6 times). Subsequent steps are completed in a sterile operating table.

[0173] 1.2 Use sterile tweezers to move the tibia and femur into another new culture dish containing PBS, then use a 20ml syringe to draw up sterile PBS, clamp one end of the tibia or femur with tweezers, cut off both ends of the bone with scissors, insert a 1ml syringe needle into the bone marrow cavity from both ends of the bone, and use a 20ml syringe filled with PBS to repeatedly flush the bone marrow up and down alternately. Pass the flushed bone marrow cells through a 70μm cell sieve while grinding, collect them into a 50mL sterile centrifuge tube, and centrifuge at 2000rpm for 4 minutes at 4℃.

[0174] 1.3 Add 1 ml of red blood cell lysis buffer (ACK) to the cell pellet, mix thoroughly to resuspend the cells, lyse at room temperature for about 90 seconds, add 10 ml of pre-cooled PBS for washing, and centrifuge at 4°C, 2000 rpm for 4 minutes.

[0175] 1.4 Resuspend the cells in RPMI 1640 medium, add β-mercaptoethanol and recombinant mouse GM-CSF (20 ng / ml) to the RPMI 1640 medium, and adjust the cell density to 2-3×10 6 / well, plated in untreated 6-well cell culture plates and incubated at 37°C, 5% CO 2 Cultivated under conditions.

[0176] 1.5 Observe the cell status. The cells should be in a semi-adherent state in clusters. Gently shake the culture plate and add culture medium every two days. On the fourth day, gently shake the culture plate, completely aspirate the culture supernatant, and immediately add 2 ml of fresh RPMI 1640 complete culture medium. Centrifuge the aspirated supernatant at 2000 rpm for 4 minutes, discard half of the supernatant and mix well, then add β-mercaptoethanol and recombinant mouse GM-CSF (20 ng / ml).

[0177] 1.6 Start laying the plates on the 6th day.

[0178] 2. Flow cytometry detection of the expression of co-stimulatory molecules in BMDC cells after forskolin treatment

[0179] 2.1 Gently pipette and collect the suspended and semi-adherent BMDC cells on day 6 into a sterile 50 ml centrifuge tube. Centrifuge at 2000 rpm for 4 min at 4°C, discard the supernatant, and add fresh BMDC culture medium to resuspend the BMDC cells to a concentration of 5 × 10 5 / ml. According to 1×10 5 / 200ul / well was plated into an untreated 48-well plate.

[0180] 2.2 The plated BMDCs were divided into two groups, and the second group was treated with Forskolin (20 μM) for 24 h.

[0181] 2.3 After the treatment, the cells were collected in a 96-well U-shaped plate, centrifuged at 4°C, 2000 rpm for 4 min, and the supernatant was discarded. The cells were washed once with PBS, centrifuged at 4°C, 2000 rpm for 4 min, and the supernatant was discarded.

[0182] 2.4 Prepare a mixed solution containing anti-MHCII, anti-CD80, anti-CD86 and zombie aqua (ZA, Live / dead) flow cytometry antibodies to perform flow cytometry surface staining on the cells.

[0183] 2.5 Detect the expression levels of BMDC co-stimulatory molecules CD80, CD86 and MHCII by flow cytometry. Zombie aqua- cells are living cells, and the expression levels of CD80, CD86 and MHCII in BMDC are reflected by detecting the percentage of positive cells and the mean fluorescence intensity.

[0184] The experimental results are as follows Figure 7 As shown in a and b, flow cytometry results showed that Forskolin could upregulate the expression of co-stimulatory molecules CD80, CD86 and MHCII on the surface of BMDC cells.

[0185] Example 7 Forskolin-treated DCs for CD8 + Effects on T cell proliferation

[0186] 1. Forskolin treatment of BMDC cells

[0187] 1.1 Bone marrow cells were cultured in a medium containing the cytokine GM-CSF for 6 days to obtain BMDCs with very weak immunogenicity.

[0188] 1.2 Gently pipette and collect the suspended and semi-adherent BMDC cells on day 6 into a sterile 50 ml centrifuge tube, centrifuge at 2000 rpm for 4 min at 4°C, discard the supernatant, add fresh BMDC culture medium and resuspend the BMDC cells to a concentration of 1×10 5 / ml. According to 1×10 4 / 100ul / well was plated into a 96-well plate.

[0189] 1.3 The plated BMDCs were divided into two groups, and the second group was treated with Forskolin (20 μM) for 24 h.

[0190] 2. Flow cytometry to obtain OT1 CD8 + T cells

[0191] 2.1 Take C57BL / 6J OT1 wild-type adult mice, kill them and soak them in 75% alcohol for 2-3 minutes for disinfection. Make an incision in the abdomen, separate the spleen with scissors, soak it in PBS buffer, and then soak and wash it with sterile PBS several times.

[0192] 2.2 Grind the spleen with the handle of a 1 ml syringe and rinse the spleen repeatedly with a 20 ml syringe filled with PBS. Pass the spleen cells through a 70 μm cell sieve while rinsing and grinding, collect them into a 50 ml sterile centrifuge tube, and centrifuge at 2000 rpm for 4 min at 4°C.

[0193] 2.3 Add 1 ml of red blood cell lysis buffer (ACK) to the cell pellet, mix thoroughly to resuspend the cells, lyse at room temperature for about 90 seconds, add 10 ml of pre-cooled PBS for washing, and centrifuge at 4°C, 2000 rpm for 4 minutes.

[0194] 2.4 Prepare a mixed solution containing flow cytometry antibodies such as anti-TCRβ and anti-CD8a, and perform flow cytometry surface staining on the cells.

[0195] 2.5 After staining, add 7AAD (Live / dead) antibody and dilute it with FACS (PBS solution containing 2% serum), and sort OT1 CD8 by flow cytometry. + T cells.

[0196] 2.6 The CD8 + T cells were centrifuged at 2000 rpm for 4 min at 4°C and added to CellTrace TM Violet for CD8 + T cell labeling and staining for 15 min; then add 10 ml PBS to terminate the staining and centrifuge at 2000 rpm for 4 min at 4°C.

[0197] 3. Forskolin-treated BMDC and CD8 + T cell co-culture

[0198] 3.1 After the forskolin treatment, the culture supernatant of BMDC was discarded and 1 ng / ml OVA peptide (257-264) was added to stimulate for 2 h. Then the stimulation supernatant was discarded and washed once with PBS. The obtained CD8 + T cells.

[0199] 3.2 Fresh RPMI 1640 complete medium to obtain CD8 + T cells were resuspended to a concentration of 5 × 10 5 / ml, supplemented with 100ng / ml human recombinant IL2. 5 / 200ul / well was re-plated into a 96-well flat-bottom plate containing BMDC cells and placed in 37°C, 5% CO 2 The cells were cultured in an incubator for 2 days.

[0200] 3.3 After proliferation, add 7AAD antibody and beads, and detect CD8 by flow cytometry + T cell proliferation peak and cell number. Figure 8 As shown in Figures ac, Forskolin treatment enhanced the expression of dendritic cells in CD8 + Example 8 Effect of Forskolin-treated DC on CD8+ Effects of T cell effector molecules IFNγ and Granzyme B

[0201] 1.CD8 + T cell stimulation

[0202] 1.1 The above Forskolin-treated BMDCs were mixed with CD8 + T cells were then treated with PMA, Ionomycin and GolgiPlug stimulators for 4 h.

[0203] 2.CD8 + T cell intracellular staining and flow cytometry

[0204] 2.1 After stimulation, CD8 + T cells were collected in a 96-well U-plate, centrifuged at 4°C, 2000 rpm for 4 min, and the supernatant was discarded. The cells were washed once with PBS, centrifuged at 4°C, 2000 rpm for 4 min, and the supernatant was discarded.

[0205] 2.2 Prepare a mixture containing zombie aqua (ZA, Live / dead) flow cytometry antibody and perform flow cytometry surface staining on cells.

[0206] 2.3 Centrifuge the cells at 4°C, 2000 rpm for 4 min, discard the supernatant, add fixative and fix on ice for 30 min.

[0207] 2.4 Use the punching solution to prepare a flow cytometry antibody mixture containing anti-IFNγ and anti-Granzyme B for intracellular staining of cells.

[0208] 2.5 Detection of CD8 by flow cytometry + Expression levels of IFNγ and anti-Granzyme B in T cells.

[0209] The experimental results are as follows Fig. 9 As shown in a and b, flow cytometry analysis showed that Forskolin treatment could enhance the expression of CD8 + Expression of IFNγ and anti-Granzyme B in T cells.

[0210] 3.BMDC and CD8 + IFNγ secretion in T cell co-culture system

[0211] Charge BMDC and CD8 + The cell culture supernatant after 48 h of T cell co-culture was used to detect the secretion of IFNγ in the co-culture system using a mouse IFNγ ELISA detection kit.

[0212] The results are as follows Fig. 9As shown in c, Forskolin treatment can enhance CD8 + Secretion of IFNγ by T cells.

[0213] Example 9 Forskolin-treated DCs for CD8 + Effect of T cell killing ability

[0214] 1. Treatment of Tumor Cells

[0215] 1.1 When the Hepa1-6BL cell density reaches 80%-90%, cell passage is performed in a clean bench as follows: discard the cell culture supernatant, add 3 ml of sterile PBS buffer to wash the cells, discard the PBS, add 0.5 ml of 0.25% EDTA trypsin digestion solution to completely cover the bottom of the cell culture dish, and place it in a 37°C, 5% CO 2 Incubator with 4% paraformaldehyde and digest for 1 min. Observe the cell morphology under an inverted microscope. When some cells fall off or the cell morphology changes, add 2 ml of complete culture medium to the culture dish and blow the cells at the bottom of the culture dish to make them fall off completely. Transfer the cells to a centrifuge tube and centrifuge at 350 g for 4 min at 4°C. Discard the supernatant.

[0216] 1.2 The Hepa1-6BL cells obtained by centrifugation were labeled with CFSE and stained for 15 minutes; then 10 ml of PBS was added to terminate the staining and centrifuged at 2000 rpm for 4 minutes at 4°C.

[0217] 1.3 Add fresh RPMI 1640 complete medium and resuspend the CFSE-labeled Hepa1-6BL cells to a concentration of 2×10 5 / ml. According to 2×10 4 / 100ul / well re-plate into 96-well flat-bottom plates and place in 37°C, 5% CO 2 cultured in an incubator.

[0218] 2. Effect of Forskolin on BMDC-mediated CD8 + The killing ability of T cells on Hepa1-6BL hepatocellular carcinoma cells

[0219] 2.1 BMDC and CD8 + CD8 T cells proliferated in co-culture system for 48 h + T cells were collected in a 1.5 ml centrifuge tube, centrifuged at 2000 rpm for 4 min at 4°C, and the supernatant was discarded.

[0220] 2.2 Add fresh RPMI 1640 complete medium to CD8 + T cells were resuspended to a concentration of 5 × 10 5 / ml. According to 1

[0221] ×10 5 / 200ul / well was added back into the 96-well flat-bottom plate containing Hepa1-6BL cells and placed in 37°C, 5% CO 2 Incubate in an incubator for 18 h.

[0222] 2.3 After the co-culture, the cells were digested with trypsin, collected in a 96-well U-shaped plate, centrifuged at 2000 rpm for 4 min at 4°C, and the supernatant was discarded.

[0223] 2.4 Prepare Annexin V flow cytometry antibody solution with Annexin V buffer and perform flow cytometry staining on the cells for 15 minutes.

[0224] 2.5 Detection of CD8 by flow cytometry + The killing ability of T cells on Hepa1-6BL cells.

[0225] The experimental results are as follows Fig.10 As shown in a and b, flow cytometric analysis showed that Forskolin treatment could enhance BMDC-mediated CD8 + T cells kill Hepa1-6BL liver cancer cells.

[0226] Example 10 Forskolin-treated dendritic cells inhibit the growth of orthotopic hepatocellular carcinoma in mice

[0227] Adoptive transfer of mGluR4-deficient dendritic cells inhibited the growth and spontaneous lung metastasis of orthotopic hepatocellular carcinoma in tumor-bearing mice. 5 Hepa1-6BL tumor cells were hydrodynamically injected into C57BL / 6WT mice via the tail vein, with 6-9 mice in each group. On days 3, 9, and 15 relative to tumor cell inoculation, BMDCs (3×10 6 ). The mice were sacrificed on day 21, and the livers were collected for tumor burden quantification.

[0228] The specific steps are as follows:

[0229] 1. Preparation of Tumor Lysate

[0230] 1.1 When the Hepa1-6BL cell density reaches 80%-90%, collect the Hepa1-6BL cells, resuspend them in fresh RPMI1640 complete medium and freeze-thaw them 6 times (-80°C for 30 min, thaw in a 37°C water bath).

[0231] 2. Preparation of Dendritic Cell Vaccine

[0232] 2.1 Gently pipette and collect the suspended and semi-adherent BMDC cells on day 6 into a sterile 50 ml centrifuge tube. Centrifuge at 2000 rpm for 4 min at 4°C, discard the supernatant, and add fresh BMDC culture medium to resuspend the BMDC cells to a concentration of 2×10 6 / ml. According to 4×10 6 / 2ml / well was plated into untreated 6-well plates.

[0233] 2.2 The plated BMDCs were divided into two groups, and the second group was treated with Forskolin (20 μM) for 24 h.

[0234] 2.3 The tumor cell lysate obtained above was added to BMDCs at a ratio of 1:1 tumor cell:BMDC (approximately one tumor cell lysate per BMDC). After pulsing with tumor lysate for 16 h in growth medium containing BMDCs, the cells were harvested, washed thoroughly with sterile PBS, and resuspended in PBS.

[0235] 3. Construction of Mouse Tumor Model

[0236] 3.1 Construction of liver carcinoma in situ in hepatocellular carcinoma mice: When the density of Hepa1-6BL cells reaches 80%-90%, cell passage is performed in a clean bench as follows: discard the cell culture supernatant, add 3 ml of sterile PBS buffer to wash the cells, discard the PBS, add 0.5 ml of 0.25% EDTA trypsin digestion solution to completely cover the bottom of the cell culture dish, put it in a 37°C, 5% CO2 incubator, digest for 1 min, observe the cell morphology under an inverted microscope, if some cells fall off or the cell morphology changes, add 2 ml of complete culture medium to the culture dish, blow the cells at the bottom of the culture dish to make them fall off completely, transfer the cells into a centrifuge tube, centrifuge at 4°C, 350g for 4 min, and discard the supernatant.

[0237] 3.2 Add the Hepa1-6BL cells obtained by centrifugation into fresh RPMI 1640 basal medium and resuspend the cells to a concentration of 3.3×10 5 / ml.

[0238] 3.3 According to 5×10 5 Hepa1-6BL / 1.5ml / mouse was injected into C57BL / 6J wild-type adult mice through the tail vein by high-pressure water injection. The mice were divided into BMDC control group and Forskolin BMDC treatment group.

[0239] 4. Injection of DC Vaccine into Tumor-bearing Mice

[0240] 4.1 After the pulse is over, gently pipette to collect the suspended and semi-adherent BMDC cells on day 6 into a sterile 50 ml centrifuge tube, centrifuge at 4°C, 2000 rpm for 4 min, discard the supernatant, add fresh RPMI 1640 basal medium and resuspend the BMDC cells to a concentration of 4×10 7 / ml.

[0241] 4.2 On days 3, 9, and 15 after tumor injection, 200 μl of the 2×10 6 Antigen-loaded BMDCs.

[0242] 4.3 At the end of the 21st day of the mouse tumor-bearing experiment, the mouse body weight, liver tumor weight, liver tumor nodules, serum and spontaneous lung metastasis were counted.

[0243] 5. Detection of serum AST and ALT in tumor-bearing mice

[0244] 5.1 At the end of the 21st day of the experiment, blood samples were collected from mice with orthotopic liver tumors. The samples were stored at room temperature and centrifuged at 3000 rpm for 10 min to obtain mouse serum.

[0245] 5.2 The levels of alanine aminotransferase (ALT), also known as alanine aminotransferase (GPT) activity assay kit and aspartate aminotransferase (AST), also known as aspartate aminotransferase (GOT) assay kit were used to measure the levels of alanine aminotransferase and aspartate aminotransferase in serum aliquots. The above biochemical parameters were tested according to the manufacturer's instructions.

[0246] The results showed that: a high-pressure liver orthotopic tumor model was constructed, and dendritic cell vaccine was adopted by tail vein. Fig.11 As shown in (ac), the growth of orthotopic hepatocellular carcinoma tumors and spontaneous lung metastasis in mice adoptively treated with Forskolin-treated BMDCs were significantly inhibited.

[0247] The mouse serum was further analyzed and the results were as follows Fig.12 As shown, the levels of AST and ALT in the serum of mice adoptively treated with Forskolin-derived BMDC cells were significantly reduced, indicating that adoptive treatment of Forskolin-derived BMDCs reduced the degree of liver damage in mice.

[0248]

[0249] Forskolin is an adenylate cyclase activator that can promote the production of intracellular cyclic adenosine monophosphate (cAMP) by activating adenylate cyclase. cAMP is an important second messenger in cells and plays a key role in the signal transduction process of various cells. Especially in immune cells, the activation of the cAMP signaling pathway is of great significance for the functional regulation of cells.

[0250] The results of Examples 6-10 show that the dendritic cell vaccine treated with AC activator Forskolin can significantly inhibit the growth of orthotopic liver cancer in C57BL / 6J wild-type mice. The vaccine is simple to prepare, has clear ingredients, can effectively and quickly promote dendritic cell maturation and antigen presentation, and induce the proliferation, activation and function of cytotoxic T lymphocytes (CTLs).

Claims

1. A dendritic cell, characterized in that: The dendritic cells are mGluR4-deficient dendritic cells or forskolin-treated dendritic cells. Preferably, the dendritic cells are GM-CSF-induced mGluR4-deficient dendritic cells or GM-CSF-induced forskolin-treated dendritic cells.

2. The dendritic cell according to claim 1, characterized in that The amino acid sequence of the mGluR4-deficient dendritic cell is shown in SEQ ID No.1 or SEQ ID No.

2.

3. The dendritic cell according to claim 1, characterized in that GM-CSF-induced forskolin-treated dendritic cells were prepared as follows: Forskolin was added to the GM-CSF differentiated dendritic cell culture system and cultured for 24-48 hours; The mass volume concentration of GM-CSF is 15-25 ng / ml; the molar concentration of forskolin is 15-25 μM.

4. A dendritic cell vaccine, characterized in that: The dendritic cell vaccine comprises dendritic cells loaded with antigens, the antigens are liver cancer cell lysates, and the dendritic cells are the mGluR4-deficient dendritic cells or forskolin-treated dendritic cells according to claim 1.

5. Use of the dendritic cell vaccine according to claim 4 in the preparation of anti-tumor drugs.

6. Use of the dendritic cells according to any one of claims 1 to 3 in promoting the maturation and function of dendritic cells.

7. An immune adjuvant, characterized in that The immune adjuvant is forskolin.

8. Use of the immune adjuvant according to claim 7 in the preparation of dendritic cell vaccines.

9. Application of mGluR4 as a tumor marker in diagnosing tumors and assessing tumor prognosis.

10. Application of mGluR4 as a drug target in the development, design, research and screening of anti-tumor drugs.

Images