CN112274534A

CN112274534A - Application of ATPIF1 gene-knocked-down dendritic cells in tumor prevention and treatment

Info

Publication number: CN112274534A
Application number: CN202011133221.8A
Authority: CN
Inventors: 钟根深; 张姣姣; 叶建平; 王辉
Original assignee: Xinxiang Medical University
Current assignee: Xinxiang Medical University
Priority date: 2020-10-21
Filing date: 2020-10-21
Publication date: 2021-01-29

Abstract

The invention relates to the field of biotechnology and medicine, and provides a Dendritic Cell (DC) with ATPIF1 gene knockdown expression. Through knocking down ATPIF1 gene in dendritic cell, dendritic cell has raised antigen taking, processing and presenting capacity in immune process, and can activate initial T cell effectively, raise T cell effect and strengthen the killing effect of T cell on tumor cell. The ATPIF1 knockdown expressed dendritic cells have potential application value in the aspect of preparing immunotherapy drugs for tumors.

Description

Application of ATPIF1 gene-knocked-down dendritic cells in tumor prevention and treatment

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a method for enhancing the antigen processing and presenting capacity of dendritic cells and application of the dendritic cells obtained by the method in tumor immunotherapy drugs.

Technical Field

Dendritic Cells (DCs) are the most powerful professional Antigen Presenting Cells (APCs) of the body, and can efficiently take, process and present antigens, immature DCs have strong migratory capacity, and mature DCs can effectively activate initial T cells, and are central links in the initiation, regulation and maintenance of immune responses. Dendritic cells are the strongest antigen presenting cells in vivo known at present, and can be classified into mature DCs (mature DCs, mDCs) and immature DCs (immure DCs, imDCs) in terms of degree of differentiation and maturation state. Immature DCs exist in non-lymphoid tissues throughout the body and have a strong ability to take up processed antigens, and once they develop into mature DCs, the ability to capture antigens is lost, becoming antigen presenting cells that activate resting T cells to generate a primary immune response. In the tumor environment, dendritic cells take up, process and present antigens, are potent activators of T cell immune responses (clear target tumor cells), recognize and phagocytose tumor or viral antigens, which are processed to deliver the antigens to the surface of DC cell membranes. Subsequently, the DC cells carrying the antigenic information are contacted with naive T cells, the antigenic information is transferred to the T cells, the T cells are stimulated to be activated and expanded, and a large number of T cells which specifically recognize tumor cells or virus infected cells are generated.

Mitochondria are important pivotal junctions in cell physiology and play important roles in controlling energy production, intermediary metabolism, cell cycle progression, the execution of cell death and signaling. Mitochondrial dysfunction such as mitochondrial membrane structural abnormality, respiratory chain inhibition, and reduction of related enzyme activity can reduce mitochondrial membrane potential, open mitochondrial membrane permeability transition pore (mPTP), and ATP synthesis disorder, which can cause mitochondrial damage and intracellular ATP level imbalance. ATPase inhibitor 1 (ATPIF 1) is a regulatory subunit of mitochondrial ATP synthase, and ATPIF1, when bound to ATP synthase, inhibits ATP synthesis under normal conditions and ATP hydrolysis under stress conditions, thereby affecting intracellular ATP levels. Colon, lung, breast and ovarian cancers all express high levels of IF1 compared to normal tissues, an increase that accounts for a decrease in the oxidative phosphorylation levels of tumor cells and an increase in anaerobic glycolysis. Lactic acid derived from tumor cells in the tumor microenvironment is an important factor for regulating the phenotype and the function of the DC, and the immune escape of the tumor cells is promoted. Michaud et al found that chemotherapy-induced autophagy in mice can lead to the release of adenosine 5' -triphosphate (ATP) by tumor cells and thereby activate an anti-tumor immune response. And it was confirmed that autophagy-dependent (extracellular) ATP recruits dendritic cells (dendritic cells) into the tumor and activates T cell response to the tumor cells.

Malignant tumor patients have low immunity and lack of powerful anti-tumor immune response due to weak antigenicity of tumor cells and low function of antigen presenting cells, so that tumor antigens cannot be effectively presented to lymphocytes. Therefore, how to effectively induce the anti-tumor immune effect is a very critical issue. The invention tries to discuss the antigen processing and presenting capability of the ATPIF1 knockout enhanced dendritic cells from a cell energy metabolism path, and provides a new strategy and a scientific basis for clinical tumor adoptive immunotherapy.

Disclosure of Invention

The invention aims to provide a dendritic cell with enhanced antigen processing and presenting capacity, which exerts the effect of enhanced anti-tumor immune response by knocking down ATPIF1 gene and down-regulating ATPIF1 protein expression.

The invention also aims to provide the dendritic cells with ATPIF1 gene knockout, and the capacity of the dendritic cells for generating ATP is enhanced under normal culture conditions.

The purpose of the invention is realized by the following technical means: the method comprises the steps of utilizing a CRISPR-Cas9 technology to carry out gene editing, knocking down an ATPIF1 gene, or utilizing siRNA, plasmid-mediated shRNA or virus-mediated shRNA to silence ATPIF1, regulating and controlling energy metabolism reprogramming of dendritic cells, enhancing the capacities of uptake, processing and antigen presentation of the dendritic cells in the immune process, effectively activating initial T cells, improving the effect function of the T cells and enhancing the killing effect of the T cells on tumor cells.

Preferably, the antigen processing and presenting ability of the dendritic cell includes the ability to recognize and phagocytose tumor or virus antigens or protein polypeptide antigens, which are processed to deliver the antigens to the surface of the DC cell membrane.

Preferably, the dendritic cells include, but are not limited to, bone marrow-derived dendritic cells, such as peripheral blood-derived dendritic cells.

The dendritic cell with the ATPIF1 gene knocked out is higher than a normal dendritic cell in aspects of ATP level generation, cell coculture with T cells, IFN-gamma secretion amount in culture supernatant, tumor cell growth inhibition effect and the like, and has higher killing activity on corresponding cancer cells. The important function of the dendritic cells with the ATPIF1 gene knocked down in the anti-tumor immune response process is highlighted, and a new regulating target point is provided for enhancing the anti-tumor effect of the dendritic cells.

Drawings

FIG. 1 shows wild type mouse and ATPIF1^-/-Inducing and culturing a protein immunoblot image of mature dendritic cells in vitro by using a mouse;

FIG. 2 shows wild type mouse and ATPIF1^-/-After FITC-OVA is added into mature dendritic cells after the mice are induced and cultured in an external induction way, detecting a contrast graph of FITC-OVA uptake of the dendritic cells by flow cytometry;

FIG. 3 shows wild type mouse and ATPIF1^-/-Mouse in vitro induction culture mature dendritic cells, after FITC-OVA is added, the H2K on the surface of the dendritic cells is detected by flow cytometry^bComparative plot of/SIINFEKL;

FIG. 4 shows wild type mouse and ATPIF1^-/-After the mature dendritic cells are cultured by in vitro induction of mice and incubated by cancer cell lysate of breast cancer cells 4T1 or melanoma cells B16 of the mice, the mature dendritic cells are added with wild type mouse CD3⁺After the T cells are co-cultured for 48, the inhibition effect of the luciferase detection T cells on the cancer cells is compared under the co-culture of different T cells and the cancer cells;

FIG. 5 is a comparison graph of IFN- γ content in supernatants of co-cultured cells measured by ELISA after co-culture of T cells and cancer cells at different ratios;

FIG. 6 shows wild type mouse and ATPIF1^-/-Inducing and culturing ATP level of mature dendritic cells in vitro by the mice;

Detailed Description

Example 1:

experimental animals: all mice (8-12 weeks old) were bred and supplied in the laboratory, ATPIF1^-/-The mouse is obtained by knocking out ATPIF1 gene by CRISPR/Cas9 technology, and the control group WT mouse is ATPIF1^+/-Matched and littermate ATPIF1^+/+Normal mice. The mice are bred in an SPF-level environment for 12 hours with alternating light and shade, the ambient temperature is 22-26 ℃, the humidity is 40% -60%, the mice are fed with low-fat feed (fat = ≥ 4% kcal), and the mice can freely eat and drink water.

Extraction, induction and culture of mouse Bone Marrow Dendritic Cells (BMDCs)

1. Obtaining mouse bone marrow cells

1) 2 mice (8-12 weeks old) of wild type mice and ATPIF 1-/-mice are killed by cervical dislocation, all femurs (femurs) and tibias (tibias) are taken out by operation, and muscle tissues around the bones are removed to the greatest extent by scissors and tweezers and are put into precooled sterile PBS; note: does not damage the bone;

2) moving the bone into a super clean bench, and washing with sterile PBS for 3 times;

3) moving the bone into another new culture dish containing sterile PBS, shearing off two ends of the bone by using scissors, extracting the PBS by using an injector, respectively inserting a needle into a marrow cavity from two ends of the bone, and repeatedly washing out the bone marrow into the culture dish until the bone is completely whitened;

4) collecting bone marrow suspension, and filtering with 200 mesh nylon net to remove small pieces and muscle tissue;

5) centrifuging the filtrate at 1200rpm for 5min, and removing the supernatant;

6) adding 3 ml of erythrocyte lysate into the bone marrow suspension of each mouse, re-suspending the cells, and incubating for 3-5 min on ice;

7) adding 10ml PBS to neutralize the action of the lysate, then centrifuging for 5min at 1200rpm, and removing the supernatant;

8) PBS was washed 1 time, and then cells were resuspended in RPMI 1640 culture medium containing 10% FBS, to which mouse bone marrow cells were obtained;

2. induced differentiation of BMDCs

1) After counting the bone marrow cells of the mouse obtained in step 1, the cell concentration was adjusted to 1X10 using 10% FBS-containing RPMI 1640 complete medium⁶/ml;

2) Spreading to 10cm culture dish, adding 10ml cells per dish, adding recombinant mouse GM-CSF (10 ng/ml) and IL-4 (10 ng/ml), culturing at 37 deg.C, 5% CO2 culture box, and culturing for 0 day;

3) gently shaking the culture plate every 2 days, then replacing half of the fresh culture solution, and supplementing the cell factors;

4) cells at day seven were already BMDCs, but not as mature;

3. complete maturation of BMDCs

Note: the BMDC obtained in the step 2 is not completely mature DC, and if the mature DC is obtained, LPS, CD40L or TNF-a and the like are required to be induced;

1) centrifuging the BMDC obtained in the step 2 at 1200rpm for 5min, and discarding the supernatant;

2) resuspend pellet with RPMI complete medium containing recombinant mouse GM-CSF (10 ng/ml) and IL-4 (10 ng/ml) and add maturation inducer LPS (1. mu.g/ml);

3) culturing at 37 deg.C in 5% CO2 incubator for 2 days;

4) collecting suspension cells and loosely adherent cells as mature dendritic cells, wherein the number of cells obtained from each mouse is about 2 × 10⁷。

Western blotting technique

1) Preparation of protein samples: collecting the mature dendritic cells cultured in example 1, and adding a protein lysate;

2) preparation of Polyacrylamide gel (SDS-PAGE): configuring 15% SDS-PAGE;

3) sample adding electrophoresis: after the BCA protein is quantified, 30 mu g of heat-denatured protein is added into each well;

4) film transfer: rotating the membrane for 45min at 200 mA;

5) and (3) sealing: 5 percent of skimmed milk powder is sealed for 2 hours at 37 ℃;

6) primary antibody incubation: adding ATPIF1 monoclonal antibody (Abcam, cat # ab 110277)

7) And (3) secondary antibody incubation: goat anti-mouse secondary antibody labeled with HRP

8) And (3) developing: ECL luminescence and color development.

As a result: as shown in FIG. 1, it is a wild type mouse and ATPIF1^-/-The detection of ATPIF1 protein of dendritic cells matured by in vitro induction culture of mice verifies that the in vitro induction culture of miceThe mature dendritic cell ATPIF1 knockout was successful.

Detection of dendritic cell uptake and antigen presentation Capacity

1. Preparation of FITC-OVA protein

1) The crosslinking reaction was ultrafiltered three times with OVA protein (concentration 2 mg/ml) at 4 ℃ to a pH of 9.0. The preparation method of the crosslinking reaction liquid comprises the following steps: 7.56 g NaHCO₃，1.06 g Na₂CO₃Adding 7.36 g of NaCl, and adding water to a constant volume of 1 liter;

2) FITC was dissolved in DMSO at a concentration of 1 mg/ml. FITC used for each crosslinking is prepared fresh and protected from light;

3) according to P: f (protein: FITC) =1 mg: slowly adding FITC into the antibody solution at a ratio of 150 mu g, and gently shaking while adding to uniformly mix the FITC and the antibody until the mixture reacts for 12 hours in a dark place of a small flat shaking table at 4 ℃ by gentle shaking;

4) adding 5 mol/L NH₄Cl to a final concentration of 50 mmol/L, and terminating the reaction at 4 ℃ for 2 hours;

5) the cross-linker was ultrafiltered in PBS for more than four times until the dialysate was clear. Obtaining FITC-labeled OVA protein with the concentration of 2mg/ml, and then diluting the protein to the used concentration by using cell culture solution;

2. taking wild type mouse and ATPIF1 ^-/-2 mice each, the dendritic cells obtained were plated on 12-well plates (2X 10 wells per well) according to the procedure described in "extraction, induction and culture of mouse Bone Marrow Dendritic Cells (BMDC)" in example 1⁶One) and adding FITC-OVA prepared in the step 1 with the concentration of 10 mu g/ml, 20 mu g/ml and 40 mu g/ml, and then incubating for 4 hours;

1) collecting cells after 4h, at 4 ℃ for 10min at 1500rpm, and discarding the supernatant;

2) adding 1mL of PBS to resuspend the cells, mixing uniformly, collecting the cells into a 1.5mL EP tube, centrifuging at 400g for 5 min;

3) discarding the supernatant, adding 1mL PBS to wash the cells, centrifuging again at 400g for 5min, and discarding the supernatant;

4) add 100ul PBS to resuspend the cells in flow tubes, adding CD11c-APC and H2K per tube ^b1. mu.l each of SIINFEKL-PE antibody; after mixing, the mixture is at room temperatureIncubating for 30min in dark;

5) after the incubation is finished, centrifuging at 4 ℃ for 5min at 400 g;

6) discarding the supernatant, adding 1mL PBS to wash the cells, centrifuging again at 4 ℃ for 5min at 400g, and then discarding the supernatant;

7) the cells were resuspended in 300. mu.L of 1 XPBS, flow cytometric experiments were performed using a flow cytometer, and analyzed using FlowJo software, with three replicates for all wells.

As a result: FIG. 2 shows wild type mouse and ATPIF1^-/-After FITC-OVA is added into mature dendritic cells after the mice are induced and cultured in an external induction way, detecting a contrast graph of FITC-OVA uptake of the dendritic cells by flow cytometry; FIG. 3 shows wild type mice and ATPIF1^-/-Mice induced culture mature dendritic cells in vitro after FITC-OVA is added, and the surface H2K of the dendritic cells is detected by flow cytometry^bComparative plot of/SIINFEKL. From FIGS. 2 and 3, ATPIF1 can be seen^-/-Uptake of OVA-FITC after FITC-OVA addition to dendritic cells and dendritic cell surface H2K^bThe expression of SIINFEKL is obviously higher than that of wild dendritic cells, which indicates that the ATPIF1 knockout enhances the uptake and antigen presentation capacity of the dendritic cells, and also indicates that the ATPIF1 knocked down dendritic cells have potential value in enhancing the anti-tumor immune response.

Detection of killing Activity on cancer cells after Co-culture of dendritic cells and T cells after cancer cell lysate bombardment

1. Taking wild type mouse and ATPIF1 ^-/-2 mice each, the dendritic cells obtained were plated on 12-well plates (2X 10 wells per well) according to the procedure described in "extraction, induction and culture of mouse Bone Marrow Dendritic Cells (BMDC)" in example 1⁶One)

2. B16 and 4T1 cells were cultured in mice, respectively. Respectively collecting 1000 ten thousand B16 and 4T1 cells, washing with PBS after centrifugation, respectively adding 200 mul sterile PBS buffer solution, freeze-thawing at-80 deg.C and ice bath for 5 times, centrifuging at 10000 g for 30min, collecting supernatant, and subpackaging and freezing;

3. the dendritic cells cultured to the seventh day in the step 1 were added with 10. mu.l of the collected cell lysate (4T 1 or B16 cell lysate) for antigen impact, and 24 hours later, T cells isolated from the spleen of a wild-type mouse were added, and the T cell isolation step was as follows:

4. isolation of mouse spleen T cells:

1) taking 4 cell culture dishes of 5cm, adding 5mL of PBS (0.1% BSA + 0.6% sodium citrate) preheated to room temperature into each cell culture dish, respectively placing a cell filter screen into each cell culture dish, taking 4 wild mice, killing the mice, taking out spleens, respectively placing the spleens into the filter screens, and grinding tissues by using a disposable syringe to push a handle end;

2) transferring the ground spleen cell suspension into a 15ml centrifuge tube by using a pipette, filling the centrifuge tube with cold PBS, and centrifuging the centrifuge tube at 1300rpm for 10 minutes;

3) the supernatant was discarded, and the cell suspension was resuspended in 15ml of cold PBS and centrifuged for 10 minutes;

4) repeating the above steps, and washing for 3 times;

5) each spleen was resuspended pellet with 5ml of red blood cell lysis buffer, incubated on ice for 5 minutes with occasional shaking;

6) the reaction was stopped by diluting the lysis buffer with 20-30ml of 1 × PBS, centrifuging to remove the supernatant, and resuspending in 5ml of PBS;

7) counting the cells;

5. magnetic bead sorting of T cells: a mouse T cell isolation kit (cat # 11413D) from Thermofeisher was purchased, and CD3 was isolated according to the procedure of the kit instructions⁺A T cell;

6. co-culturing the T cell suspension obtained in the step 5 and the dendritic cells stimulated by the tumor antigen obtained in the step 3

1) According to 4X 10⁶Number of cells/well the T cells purified in step 5 were added to the dendritic cells in step 3 (12 wells total, 2X 10 cells per well)⁶One);

2) collecting T cells after 2 days, at 4 ℃, 10min and 1500rpm, and discarding the supernatant;

3) adding 1mL of PBS to resuspend the cells, mixing uniformly, collecting the cells into a 1.5mL EP tube, centrifuging at 400g for 5 min;

4) after discarding the supernatant, 1mL of PBS was added to resuspend the cells;

7. co-culture of T cells and cancer cells

1) Spreading 4T-1 and B16 containing luciferase plasmids on a 96-well plate according to the number of 5000 per well, and culturing overnight;

2) the T cells in step 6 were differentiated from the cancer cells in terms of the ratio (T cells: cancer cells =5:1, 1:1 and 1: 5) were added to 4T-1 or B16 (3 duplicate wells each) grown in 96-well plates for 48h of co-culture;

3) collecting co-culture supernatant for later use;

4) adherent cells after discarding the supernatant were added with a luciferase assay kit purchased from Shanghai Biyuntian Biotech Co., Ltd (cat #: RG 005), according to the experimental steps of the specification, adding 100 mul of lysis solution into each well of a 96-well plate, adding 100 mul of luciferase reaction reagent after 15min, detecting the fluorescence intensity of each well by adopting a multi-wavelength microplate reader, analyzing and comparing the luciferase intensity under the co-culture of T cells and cancer cells with different ratios, and repeating all detection wells for three times.

The results show that: after the dendritic cells and the T cells are co-cultured after being impacted by 4T1 and B16 cancer cell lysate, the T cells are activated and further activated with the luciferase-containing plasmid 4T1^Luc+Or B16^Luc+After co-cultivation. In different T cells: at the rate of cancer cells, the growth of tumor cells can be inhibited, especially T cells: cancer cells at 5: 1; while ATPIF1^-/-The T cells co-cultured by the dendritic cells have higher tumor growth inhibition capacity, and have significant difference (P) compared with the T cells co-cultured by wild dendritic cells<0.001) and basically has a 20% difference in the lysis rate of cancer cells, which shows that the ATPIF1 can remarkably enhance the presenting capability of the ATPIF1 to antigen after being knocked down, thereby increasing the stimulation to T cells and further enhancing the killing activity of the T cells to corresponding cancer cells.

Enzyme-linked immunosorbent assay (ELISA) was performed to detect IFN-. gamma.expression in the co-cultured cell culture supernatant (supernatant from step 7) of example 5).

IFN-gamma content was determined according to the ELISA kit procedure for IFN-gamma detection (kit purchased from Thermofisiher, cat # 88-7314-22) and analyzed for comparison of IFN-gamma content in cocultures of different ratios of T cells and cancer cells. The result shows that the T cells stimulated by dendritic cells with the expression knocked down by ATPIF1 can secrete more IFN-gamma after being cultured with the cancer cells, and the two cells have a significant difference (P < 0.001) compared with the T cells stimulated by wild type WT dendritic cells. Indicating that ATPIF1 knockdown dendritic cells can activate more active CD3+ T cells.

Detection of dendritic cell ATP levels

1) Taking wild type mouse and ATPIF1 ^-/-1 mouse, the dendritic cells obtained were collected in a 1.5mL EP tube, centrifuged at 1000 rpm at 4 ℃ for 5min, centrifuged, and the supernatant was discarded, 100. mu.L of white Cell Lysis Buffer was added, mixed, and lysed on ice for 30min according to the procedure described in "extraction, induction and culture of mouse Bone Marrow Dendritic Cells (BMDC)" in example 1. Then centrifuging for 10min at 4 ℃ at the rotating speed of 12000 g, and taking the supernatant to be the sample to be detected. Note that the whole process was operated on ice;

2) preparing ATP standard substances and ATP detection working solution with different concentrations according to the instructions of an ATP Determination Kit (A22066) Kit;

3) adding 10 μ L of diluted ATP standard with concentration of 0, 1nM, 5nM, 10nM, 0.1 μ M, 0.5 μ M, 1 μ M, 5 μ M into light-shielding 96-well plate; after the sample to be tested was diluted 10-fold with 1 × PBS, 10 μ L of the diluted sample was added to a light-shielding 96-well plate. The whole process is operated on ice, and all detection holes are provided with three auxiliary holes;

4) under the environment of keeping out of the sun, adding 90 mu L of ATP detection working solution into each hole, and then detecting the light absorption values of the standard substance and the sample at 560 nm by using an enzyme-labeling instrument;

5) and (4) calculating the calculation formula of the ATP concentration according to the concentrations and light absorption values of the ATP standard substances with different concentrations. Substituting the light absorption value of the sample into a formula to calculate the ATP levels in different samples;

6) detecting the protein concentration of each sample by using a BCA kit, and dividing the ATP level by the protein concentration to obtain the final ATP/protein concentration for comparison;

as a result: FIG. 6 shows wild type mice and ATPIF1^-/-Detection of ATP levels in mice induced cultured mature dendritic cells in vitro can show that ATPIF1 knockout dendritic cells produce more ATP than wild type dendritic cells. Therefore, it is suggested that an increase in ATP may be one of the mechanisms for enhancing the antigen presenting ability of dendritic cells.

Claims

1. Application of ATPIF1 gene-knocked-down dendritic cells in preparation of tumor prevention and treatment medicines.

2. Use according to claim 1, characterized in that: the dendritic cells are bone marrow-derived dendritic cells or peripheral blood-derived dendritic cells.

3. Use according to claim 1, characterized in that: the medicine is used for preventing and actively treating tumors.

4. Use according to claim 1, characterized in that: the method for knocking down the dendritic cell ATPIF1 gene comprises the steps of carrying out gene editing by using a CRISPR-Cas9 technology, and using siRNA, plasmid-mediated shRNA or virus-mediated shRNA to silence ATPIF 1.