An in vitro treatment method for improving Th9 cell differentiation efficiency

Abstract

The present invention discloses an in vitro treatment method for improving the differentiation efficiency of Th9 cells, and relates to the field of biomedicine technology. + When T cells differentiate into Th9 cells, sorafenib is used to treat CD4 + T cells, and found that sorafenib can significantly improve the differentiation efficiency of Th9 cells in vitro, indicating that sorafenib can amplify Th9 cells with anti-tumor effects in vitro, providing new ideas and new approaches for the ACT treatment of clinical tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an in vitro treatment method for improving Th9 cell differentiation efficiency. Background Art

[0002] As a cutting-edge branch in the field of cancer immunotherapy, adoptive cell therapy (ACT) has made significant progress in tumor treatment in recent years, providing new treatment hope for many patients. The basic principle of this therapy is to activate and amplify the patient's own immune cells, especially tumor-specific T cells, in vitro, and then return these modified cells to the patient to identify and destroy tumor cells. Traditionally, ACT mainly focuses on CD8 + Cytotoxic T cells (CTLs) are key effector cells in tumor immune response, which can directly kill recognized cancer cells. + The role of T cells in anti-tumor immunity is becoming increasingly prominent, especially their subsets such as Th1 and Th17 cells, which participate in the anti-tumor process indirectly or directly by regulating immune responses, promoting the activation and maintenance of CTLs, and regulating the tumor microenvironment.

[0003] Among many CD4 + Among T cell subsets, Th9 cells are a relatively new group with great research potential. Since they were first discovered and reported in 2008, Th9 cells have attracted much attention due to their unique cytokine secretion pattern (mainly secreting IL-9, but also producing IL-21 and IL-3) and their important role in various diseases. In tumor immunology, Th9 cells have shown unique anti-tumor potential. Through the secretion of cytokines such as IL-9, they can not only directly act on tumor cells to inhibit their growth and proliferation, but also activate and regulate other immune cells, such as dendritic cells (DCs), CD8 + CTLs, natural killer cells (NK cells) and mast cells form a complex immune network that works together in the tumor microenvironment to promote the enhancement of anti-tumor immune responses.

[0004] It is particularly worth mentioning that recent studies have shown that melanoma can be completely eradicated in mouse models by reinfusing Th9 cells. This result not only verifies the effectiveness of Th9 cells in anti-tumor treatment, but also reveals that they may have significant therapeutic effects that surpass other T cell subsets (such as Th1 and Th17) in certain types of tumors (such as melanoma, which is usually considered a "cold tumor"). Cold tumors refer to tumors with less immune cell infiltration and weaker immune responses. Such tumors often respond poorly to traditional immunotherapy, and the discovery of Th9 cells provides a new strategy for the treatment of such tumors.

[0005] However, although Th9 cells have shown great therapeutic potential, how to effectively expand these cells in vitro to meet the needs of clinical ACT treatment is still a major challenge currently faced. Traditional cell expansion methods may not be able to efficiently induce and maintain the differentiation of Th9 cells, and may be accompanied by problems such as decreased cell function or deviation in differentiation direction. Therefore, the development of new and efficient Th9 cell expansion technology is of great significance to promote the application of ACT in tumor treatment.

[0006] On the other hand, sorafenib (Sora for short) as a multi-target, multi-kinase inhibitor has shown effective inhibitory effects on a variety of tumors (including hepatocellular carcinoma, renal cell carcinoma and certain types of thyroid cancer) in clinical practice. It achieves the dual effects of inhibiting tumor proliferation and angiogenesis by inhibiting key kinases on tumor cells and their blood vessels, such as serine / threonine kinases and receptor tyrosine kinases. Although sorafenib is mainly used as a monotherapy, in recent years, there have also been studies exploring the possibility of combining it with other treatments (such as immunotherapy) in order to achieve better therapeutic effects.

[0007] However, there is no report on whether sorafenib is correlated with Th9 cells. Summary of the invention

[0008] Based on the deficiencies in the prior art, the present invention provides an in vitro treatment method for improving the differentiation efficiency of Th9 cells.

[0009] Initial CD4 + T cells can be induced in vitro by TGF-β combined with IL-4 to differentiate into a type of Th9 cells that can secrete IL-9, IL-10 and IL-21. Based on this method, the applicant will further improve the differentiation efficiency of Th9 cells. The applicant screened whether the kinase inhibitors commonly used in clinical practice can improve the differentiation efficiency of Th9 cells and found that sorafenib can significantly promote the differentiation of Th9 cells.

[0010] The applicant's preliminary research data showed that sorafenib can significantly improve the differentiation efficiency of Th9 cells in vitro. Analysis of the relevant mechanism showed that sorafenib promoted Th9 cell differentiation dependent on the ERK signaling pathway.

[0011] Therefore, the Th9 cells treated and untreated with Sorafenib in vitro were infused back into the tail vein of mice with other cold tumors, such as breast cancer and osteosarcoma lung metastases. The results showed that compared with mice with breast cancer and osteosarcoma lung metastases that were infused with Th9 cells not treated with Sorafenib, the anti-tumor immunity of mice with breast cancer and osteosarcoma lung metastases that were infused with Th9 cells treated with Sorafenib was enhanced and the tumor progression was slow.

[0012] First, the present invention provides an in vitro treatment method for improving the differentiation efficiency of Th9 cells. + When T cells differentiate into Th9 cells, sorafenib is used to treat CD4 + T cells.

[0013] Preferably, the concentration of sorafenib used is 0.2-2 μM. The method further includes inducing Th9 cells, wherein the induction treatment conditions are: using 10 μg / ml anti-IFNγ, 20 ng / ml IL4 and 10 ng / ml TGF-β for plating and inducing differentiation.

[0014] The present invention also provides the use of sorafenib in preparing a drug for resisting lung metastatic tumors, wherein the sorafenib is sorafenib itself or a pharmaceutically acceptable salt.

[0015] Sorafenib is used clinically to treat unresectable hepatocellular carcinoma, advanced renal cell carcinoma, and locally recurrent or metastatic, progressive, differentiated thyroid cancer that is difficult to treat with radioactive iodine. Therefore, as a ready-made drug, sorafenib has extremely high safety in the treatment of lung metastatic tumors. At the same time, in vivo test results showed that the reinfusion of sorafenib-treated Th9 cells significantly prolonged the survival time of the mouse 4T1-OVA (mouse breast cancer cells) or K7M2-OVA (mouse osteosarcoma cells) lung metastasis tumor model. The mechanism is that sorafenib can significantly increase the expression level of protein kinase p-ERK in Th9 cells.

[0016] Preferably, the lung metastatic tumor is caused by breast cancer or osteosarcoma.

[0017] In CD4 + Sorafenib was added when T cells differentiated into Th9 cells, and the concentration of sorafenib used was 0.2~2 μM.

[0018] In vivo experiments showed that the infusion of sorafenib-treated Th9 cells significantly increased the CD8+ CD45 + The proportion of cells decreased, reduced the 4T1-OVA and K7M2-OVA tumor areas in the mouse lungs, and thus prolonged the survival time of the mouse 4T1-OVA or K7M2-OVA lung metastasis tumor model.

[0019] The present invention also provides a drug for anti-pulmonary metastatic tumor, the active ingredient of which comprises Th9 cells, wherein the Th9 cells are CD4 + The method is to add sorafenib to stimulate the differentiation of T cells into Th9 cells, wherein the sorafenib is sorafenib itself or a pharmaceutically acceptable salt thereof. The concentration of sorafenib used is 0.2-2 μM.

[0020] Preferably, the dosage form of the drug is a liquid injection. In the present application, Th9 cells are infused back into the body of mice with 4T1-OVA or K7M2-OVA lung metastasis tumor models through the tail vein. The dosage of the liquid injection is 3×10 6 Th9 cells.

[0021] In summary, the present invention found that sorafenib can significantly improve the differentiation efficiency of Th9 cells in vitro, indicating that sorafenib can amplify Th9 cells with anti-tumor effects in vitro, providing new ideas and new approaches for the ACT treatment of clinical tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 For normal C57 mice naïve CD4 + The ability of T cells to polarize toward Th9 cells, with or without sorafenib treatment in vitro, specifically the proportion of Th9 cells detected by flow cytometry, a is the figure of flow cytometry detection of Th9 cells, and b is the statistical analysis of the results of figure a. "**" indicates P < 0.005; "***" indicates P < 0.001.

[0023] Figure 2 Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression level of cytokine IL9 in the supernatant of Th9 cells. "*" indicates P < 0.05; "**" indicates P < 0.005; "***" indicates P < 0.001.

[0024] Figure 3 Real-time fluorescence quantitative analysis of Th9 cells Il9 Gene expression levels. “*” indicates P < 0.05; “**” indicates P < 0.005; “***” indicates P < 0.001.

[0025] Figure 4 For normal C57 mice naïve CD4 +The expression levels of ERK signaling pathway proteins during the polarization of T cells to Th9 cells were analyzed by western blotting (WB) with or without sorafenib treatment in vitro to detect the expression levels of p-ERK, ERK and β-Actin proteins in Th9 cells.

[0026] Figure 5 For normal C57 mice naïve CD4 + The ability of T cells to polarize toward Th9 cells, with or without sorafenib or U0126 (MEK1 / MEK2 inhibitor) treatment in vitro, specifically the proportion of Th9 cells detected by flow cytometry, a is the figure of flow cytometry detection of Th9 cells, and b is the statistical analysis of the results of figure a. ns indicates no significant difference; "*" indicates P < 0.05; "**" indicates P < 0.005.

[0027] Figure 6 The lung metastasis tumor model was induced by injection of 4T1-OVA cells into the tail vein of normal C57 mice, and Th9 cells treated or not treated with sorafenib were infused back into the tail vein or not. Specifically, the lung section tumor area percentage and survival curve of the mouse 4T1-OVA lung metastasis tumor model were detected by hematoxylin-eosin staining. a is the hematoxylin-eosin staining picture of the lung, b is the statistics of the lung section tumor area percentage for a, and c is the survival curve of the tumor mouse. "*" indicates P < 0.05; "**" indicates P < 0.005; "***" indicates P < 0.001.

[0028] Figure 7 The lung metastasis tumor model was induced by injecting K7M2-OVA cells into the tail vein of normal C57 mice, and Th9 cells treated or not treated with sorafenib were infused back into the tail vein or not. Specifically, the lung section tumor area percentage and survival curve of the K7M2-OVA lung metastasis tumor model in mice were detected by hematoxylin-eosin staining. a is the hematoxylin-eosin staining picture of the lung, b is the statistics of the lung section tumor area percentage for a, and c is the survival curve of tumor mice. "*" indicates P < 0.05; "**" indicates P < 0.005; "***" indicates P < 0.001.

[0029] Figure 8 Flow cytometry analysis of CD8 in the tumor microenvironment of mice + T cell ratio, treatment conditions: normal C57 mice were injected with 4T1-OVA cells through the tail vein to induce lung metastasis tumor model, and Th9 cells treated with or without sorafenib were infused back into the tail vein. "*" indicates P < 0.05; "**" indicates P < 0.005.

[0030] Fig. 9 Flow cytometry analysis of CD8 in the tumor microenvironment of mice +T cell ratio, treatment conditions: normal C57 mice were injected with K7M2-OVA cells through the tail vein to induce lung metastasis tumor model, and sorafenib-treated or untreated Th9 cells were infused back into the tail vein or not. "**" indicates P < 0.005; "***" indicates P < 0.001. DETAILED DESCRIPTION

[0031] The C57 mice used in the present invention were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0032] Example 1

[0033] T cell sorting:

[0034] (1) Take the spleen and lymph nodes of C57 mice and squeeze them with a syringe piston to make a tissue suspension;

[0035] (2) Transfer to a 15 ml conical test tube and allow large aggregates to settle to the bottom of the tube or filter through a nylon filter to obtain a single-cell suspension;

[0036] (3) Centrifuge the cell suspension at 1500 rpm at 4°C for 5 min and discard the supernatant.

[0037] (4) Resuspend the sample in 2 ml PBS buffer, dilute with 10 μl 3% glacial acetic acid, and count the cells;

[0038] (5) Centrifuge the cells again, discard the supernatant, and resuspend the cells in sorting buffer to adjust the density to 1×10 8 Pieces / ml;

[0039] (6) Use CD4 negative selection kit (EasySepTM Mouse CD4 + CD4 + T cells for subsequent experiments.

[0040] Example 2

[0041] In vitro differentiation of Th9 cells: In a 96-well plate, dilute anti-CD3 (145-2C11, Bio Xcell) and anti-CD28 (PV-1, Bio X cell) with autoclaved PBS to a final concentration of 2 μg / ml, coat the plate at 200 μl / well, and incubate at 37°C for more than 2 hours.

[0042] CD4 + T cells, 4 × 10 510 μg / ml anti-IFNγ (BE0054, Bio X cell), 20 ng / ml IL4 (130-094-061, Miltenyi Biotec) and 10 ng / ml TGF-β (130-095-067, Miltenyi Biotec). Sorafenib (HY-10201, MedChemExpress) was added or not for stimulation. The concentrations of sorafenib were 0.2μM, 0.5μM, 1μM and 2μM respectively. The cells were plated and differentiated on day 0. The cells were Th9 cells on day 4 for subsequent experiments.

[0043] Example 3

[0044] Flow cytometry: To detect the intracellular cytokine IL-9 in Th9 cells, the cells need to be treated before staining. After stimulating with 50ng / mL phorbol ester (PMA, IP1010, Solarbio), 1 μg / mL ionomycin (Ionomycin, I8800, Solarbio) and 3μg / mL Brefeldin A (00-4506-51, Thermofisher) for 4 hours, the samples are collected. The cells to be tested are washed once with PBS, centrifuged and resuspended in PBS. CD4-PE (12-0041-82, Thermofisher) flow cytometry antibody is added, incubated on ice or at 4℃ in the dark for 30 minutes, neutralized with PBS and centrifuged twice, the supernatant is discarded, and 200μl 1× flow cytometry intracellular fixation buffer (00-8222-49, Thermofisher) is added to each tube, and incubated at room temperature in the dark for 20 minutes. After neutralization with flow cytometry permeabilization buffer (00-8333-56, Thermofisher), centrifuge. After centrifugation, discard the supernatant, resuspend the cells with 100 μl 1× flow cytometry permeabilization buffer, add IL9-APC (50-8091-82, Invitrogen) flow cytometry antibody, incubate at 4°C in the dark for 30 min, add 1× flow cytometry permeabilization buffer to neutralize, and wash twice. Discard the supernatant, resuspend the cells with an appropriate amount of PBS, and perform flow cytometric analysis using a NovoCyte flow cytometer (ACEA). The data are analyzed using FlowJo software.

[0045] If you need to detect T cell surface antigens CD45 and CD8, wash the cells with PBS once, centrifuge and resuspend them in PBS. Add CD45-PB (MCD4528, Thermofisher) and CD8-APC (MHCD0805, Thermofisher) flow cytometry antibodies, incubate on ice or at 4°C in the dark for 30 min, neutralize with PBS and centrifuge twice, discard the supernatant, resuspend the cells in an appropriate amount of PBS and perform flow cytometric analysis. Figure 1 The results showed that sorafenib significantly increased IL9 + CD4 + The number of T cells (Th9 cells). Figure 5 The results showed that the addition of U0126 (0.5 μM, HY-12031A, MedChemExpress) abolished the inhibitory effect of sorafenib on IL9 + CD4 + Effect of T cell (Th9 cell) number.

[0046] Example 4

[0047] Enzyme-linked immunosorbent assay: Collect the supernatant of Th9 cells obtained in Example 2, and take the supernatant as the sample after centrifugation. Use the kit (88-8092-88, Thermofisher), balance the pre-coated plate and the required reagents to room temperature, add 350 μl of washing buffer to the well plate, let it stand for 40s, discard the supernatant and tap the well plate on absorbent paper, and repeat three times. The standard lyophilized powder is diluted with the sample diluent, and the standards of different concentrations and the prepared samples are added to the well plate at 100 μl / well, and 100 μl of sample diluent is added to the blank well, and each sample is repeated 3 times. Seal the well plate with a sealing film, incubate at 37°C for 2 h, then discard the liquid in the well, repeat washing 3 times, add 100 μl of streptavidin-horseradish peroxidase working solution to the well, seal the plate with a sealing film, and incubate at 37°C for 30 min. Discard the liquid in the well and repeat washing 3 times. Add 100 μl of tetramethylbenzidine substrate to the wells, incubate at 37°C in the dark for 15-20 min, add 50 μl of stop solution after the liquid in the well turns blue, and measure the OD value using an enzyme-labeled instrument at room temperature. Calculate the cytokine content in the sample based on the measured OD value. Figure 2 The results showed that sorafenib significantly increased the content of IL9 in the supernatant of Th9 cells, and the content increased with the increase of sorafenib concentration.

[0048] Example 5

[0049] RNA reverse transcription: Total RNA from cells treated with sorafenib (0.2 μM, 0.5 μM, 1 μM, 2 μM) and untreated cells was extracted according to the instructions of TaKaRa's RNAiso Plus. A total of 1 μg RNA was used as a template for reverse transcription and cDNA synthesis according to the instructions of the TaKaRa reverse transcription kit (#RR037A). The reverse transcription system was prepared as follows: 5 × PrimeScriptBuffer (2 μl), Oligo dT Primer (0.5 μl), Random 6 mers (0.5 μl), PrimeScript RTEnzyme Mix I (0.5 μl), Total RNA (500 ng), RNase Free H2O (filled to 10 μl), reverse transcription reaction was carried out at 37℃ for 15 min, and the enzyme activity was inactivated by heat at 85℃ for 5 s.

[0050] Real-time fluorescence quantitative PCR: The synthesized cDNA can be used as a template for the reaction. The corresponding fluorescence quantitative PCR primers are used to perform quantitative PCR amplification using SYBR Premix Ex TaqTM (#RR041A). The fluorescence quantitative PCR system is 10 μl: SYBR Mix (5 μl), cDNA template (1 μl), primer (0.6 μl), ddH2O (filled to 10 μl). The following two-step PCR amplification standard procedure is used. Step 1: pre-denaturation, 95℃ 30s; Step 2: PCR reaction (40 cycles), 95℃ 5s, 60℃ 30s.

[0051] The PCR primer sequences are as follows:

[0052] Actb -F: 5′-ATGGAGGGGAATACAGCCC-3′,

[0053] Actb -R: 5′-TTCTTTGCAGCTCCTTCGTT-3′;

[0054] Il9 -F: 5′-TCCACCGTCAAAATGCAGCTGC-3′,

[0055] Il9 -R: 5′-CCGATGGAAAACAGGCAAGAGTC-3′.

[0056] Figure 3 The results showed that sorafenib significantly increased the expression of Th9 cells Il9 The expression level of TNF-α was significantly increased with the increase of sorafenib concentration.

[0057] Example 6

[0058] Protein extraction: Th9 cells treated with 2 μM sorafenib and untreated were collected into 1.5 mL EP tubes, washed twice with PBS, and 30 μl of RIPA lysis buffer (R0020, Solarbio) containing protease inhibitor PMSF (P0100, Solarbio) was added, mixed and placed on ice for 30 min. Centrifuged at 12000 × g for 10 min at 4°C, the supernatant was taken to a new EP tube, 5× SDS Loading Buffer (P1040, Solarbio) was added, and the protein sample was prepared for later use.

[0059] Protein immunoblotting: Place the prepared gel on the electrophoresis core, pour in the electrophoresis solution (Tris 3 g / L, glycine 14.4 g / L, SDS 1 g / L), add the Protein Ladder (26616, Solarbio) and the protein samples prepared above into the wells one by one, start at 90 V, and adjust the voltage to 180 V after the Protein Ladder is separated. After the end, take out the gel, cut into appropriate sizes and soak in transfer solution (Tris 3 g / L, glycine 14.4 g / L, 20% methanol). Cut the PVDF membrane (IPVH00010, Millipore) of the corresponding size and activate it in methanol for more than 30 s. Place the transfer clamp in the order of black splint-black sponge-thick filter paper-gel-PVDF membrane-thick filter paper-black sponge-transparent splint, put it in the rack, pour in the transfer solution, and transfer at 220 mA for 90 min. The PVDF membrane was removed and incubated in 5% skim milk at room temperature on a shaker for 1 h. The membrane was washed 3 times with PBST for 5 min each time, and then incubated with the corresponding primary antibody (p-ERK, #4370; ERK, #9102; β-Actin, #4967, Cell signaling technology) at 4°C on a shaker overnight. The primary antibody was discarded the next day, the membrane was washed 3 times with PBST for 10 min each time, and the secondary antibody (#7074P2, Cell signaling technology) was incubated on a shaker at room temperature for 1 h. The secondary antibody was discarded, and the membrane was washed 3 times with PBST for 10 min each time. The membrane was gently wiped dry with absorbent paper, and ECL (4AW011-500A / B, Sizhengbai Bio) was added and exposed using an exposure instrument.

[0060] Figure 4 The results showed that sorafenib significantly increased the expression level of protein kinase p-ERK in Th9 cells.

[0061] Example 7

[0062] Mouse lung metastasis tumor model: On Day 0, 4T1-OVA and K7M2-OVA tumor cells were collected, washed twice with PBS, and counted under a microscope. The cell density was adjusted to 1×10 7 Each C57 mouse was injected with 100 μl of tumor cell suspension through the tail vein. The cells were mixed by pipetting before injection.

[0063] T cell reinfusion therapy: Th9 cells treated with 2 μM sorafenib and untreated were obtained according to the method in Example 2, resuspended in PBS, and 3×10 cells were reinfused into the tail vein of lung metastatic tumor model mice using insulin needles on Day 7 and Day 14, respectively. 6 cells or an equal volume of PBS, and observe the survival of mice every day. Figure 6 c and Figure 7 The results in c showed that the re-infusion of Th9 cells significantly prolonged the survival time of the mouse 4T1-OVA or K7M2-OVA lung metastasis tumor model; the re-infusion of Th9 cells stimulated by sorafenib significantly enhanced this effect.

[0064] Example 8

[0065] A mouse lung metastasis tumor model was established according to Example 7. The mice were treated on day 17 and the lungs were taken for H&E staining. Figure 6 a and b in Figure 7 The results in a and b show that the reinfusion of Th9 cells significantly reduced the area of ​​4T1-OVA and K7M2-OVA tumors in the lungs of mice and prolonged their survival time; the reinfusion of Th9 cells stimulated by sorafenib further enhanced this effect.

[0066] Example 9

[0067] Lung flow cytometry: A mouse lung metastasis tumor model was established according to Example 7. On day 17, mice were treated, and the lungs were removed, ground, and filtered through a filter. The filtrate was centrifuged and resuspended in 1 mL of red blood cell lysis buffer (R1010, Solarbio), allowed to stand at room temperature for 5 min, centrifuged and the supernatant discarded, and flow cytometry was performed according to Example 3. Figure 8 and Fig. 9 The results showed that the infusion of Th9 cells significantly increased the CD8 + CD45 + The proportion of cells; the reinfusion of Th9 cells stimulated by sorafenib significantly enhanced this effect.

Claims

1. An in vitro treatment method for improving the differentiation efficiency of Th9 cells, characterized in that: In CD4 + When T cells differentiate into Th9 cells, sorafenib is used to treat CD4 + T cells; The concentration of sorafenib used was 0.2–2 μM; The in vitro treatment method also includes inducing Th9 cells, and the induction treatment conditions are: using 10 μg / ml anti-IFNγ, 20 ng / ml IL4 and 10 ng / ml TGF-β for plating and inducing differentiation.

2. A method for preparing a drug for preventing lung metastatic tumors, characterized in that: The active ingredient of the drug comprises Th9 cells, and the preparation method of the Th9 cells is: In CD4 + The method is obtained by adding sorafenib to stimulate the differentiation of T cells into Th9 cells, wherein the sorafenib is sorafenib itself or a pharmaceutically acceptable salt thereof; The concentration of sorafenib used was 0.2–2 μM; The differentiation process also includes the induction of Th9 cells. The induction conditions are: using 10 μg / ml anti-IFNγ, 20ng / ml IL4 and 10 ng / ml TGF-β for plating and inducing differentiation.

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