CN104830769B - Application of Butylated Hydroxyanisole in Expansion of Central Memory T Cells in Vitro - Google Patents

Abstract

The invention provides the application that butylated hydroxy anisole is expanded in vitro to central memory t cell, the concentration of the butylated hydroxy anisole is 5 ~ 40uM, and optimum concentration is 20uM, and the central memory t cell is CD4⁺T cell.Application of the butylated hydroxy anisole in vitro in T cell culture is made public for the first time in the present invention, method safety is effectively, with low cost, and good technical support is provided for the extensive development of adoptive immunotherapy.

Description

Application of Butylated Hydroxyanisole in In Vitro Expansion of Central Memory T Cells

technical field

The invention belongs to the technical field of in vitro expansion of central memory T cells, and more specifically relates to the application of butylated hydroxyanisole in the in vitro expansion of central memory T cells.

Background technique

Butylated hydroxyanisole (English: Butylated hydroxyanisole, referred to as BHA), is an antioxidant, for two isomers 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole A mixture of anisole, BHA is primarily used as an antioxidant and preservative in food (including animal food), food packaging, cosmetics and petroleum products. In addition, it is also used for anti-oxidation of drugs, such as isotretinoin, simvastatin and so on.

Since T cells play an important role in the immune system, more and more research groups are trying to use T cell adoptive therapy to treat tumors. Central memory T cells (TCM) have the ability of self-renewal, and the response time is short, the action time is long, and the side effects on the human body are small. In recent years, several research groups have reported that after reinfusion of T cells with memory function into tumor patients, the therapeutic effect is obvious and the treatment lasts for a long time, which can reduce the pain of patients and reduce the biosafety risk of excessive cell culture in vitro. However, the relative number in the human body is relatively small, and because long-term culture of T cells in vitro will cause T cells to lose their vitality, most of the cells will differentiate into terminally differentiated effector cells, making the survival time of T cells reinfused into the body too short. It cannot produce the function of continuously killing tumor cells, so how to obtain a large number of memory T cells in vitro is a new research hotspot in cellular immunotherapy.

At present, many foreign research groups have constructed artificial antigen-presenting cells and applied them to the in vitro culture of T cells to improve the survival time of T cells for adoptive immunotherapy. However, to obtain a sufficient amount of T cells, the technical requirements are complicated. , the success rate of cultivation is low. When performing T cell therapy, the main strategy is to obtain a sufficient amount of T cells with killing function through clonal expansion first, which can be co-cultured with presenting cells such as DC, or directly stimulate cell clonal expansion with relevant tumor antigens, T cells specific for a particular tumor antigen are obtained. The T cells cultivated by this method have not undergone any genetic modification and are relatively safe. However, such cells with high tumor reactivity need to be expanded to the clinically required 10 ⁹ -10 ¹¹ therapeutic dose, and the process lasts for a long time. For cancer patients, time is also a factor that needs to be considered more. Moreover, after a long time of in vitro culture, the cells tend to age and their vigor decreases, and most of them are terminally differentiated cells, which have good effects in vitro, but the survival time in vivo is short, and the therapeutic effect is affected.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiency of the existing central memory T cell in vitro expansion technology, and to provide the application of butylated hydroxyanisole to the central memory T cell in vitro expansion.

The above object of the present invention is achieved through the following technical solutions:

The invention provides the application of butyl hydroxyanisole to the expansion of central memory T cells in vitro.

Preferably, the concentration of butylated hydroxyanisole is 5-40uM.

More preferably, the concentration of butylated hydroxyanisole is 20uM.

Preferably, the central memory T cells are CD4 ⁺ T cells.

Preferably, the method for extracting the central memory T cells is to firstly mix the peripheral blood components with PBS containing 2% BSA and 0.5% EDTA at a ratio of 1:4; then slowly add the diluted blood to the lymphocyte separation solution. Above, the ratio of the two is 1:1; centrifuge at 300×g for 30 min; carefully absorb the mononuclear cells, put them into another centrifuge tube, add 5 times the volume of PBS containing 0.5% BSA and 2% EDTA to dilute, mix After uniformity, 300×g for 15 min; repeat the previous step once; incubate CD4+ T cell negative selection primary antibody for 15 min; dilute with 10 times the volume of PBS containing 0.5% BSA and 2% EDTA, mix well, 300× g for 12 min; incubate the secondary antibody with magnetic beads for 30 min; pass through the column for cell sorting to obtain CD4+ T cells.

Preferably, the method for culturing the central memory T cells is to place 5×10 ⁵ CD4 ⁺ T cells per well in 1 mL containing 10% fetal bovine serum under the conditions of 5% CO ₂ , saturated humidity and 37°C Cultured in RPMI1640.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

The invention discloses the application of butyl hydroxyanisole in in vitro T cell culture for the first time, the method is safe, effective and low in cost, and provides good technical support for the extensive development of adoptive immunotherapy.

Description of drawings

Figure 1 is the detection of the proportion of CD4 ⁺ TCM in CD4 ⁺ T cells when the concentration of BHA is 20uM.

Figure 2 shows the effect of different concentrations of BHA on the proportional expansion of central memory CD4 ⁺ T cells.

detailed description

The present invention will be further described below in conjunction with specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

Example 1

1. Preparation of test materials

Human peripheral blood was provided by Guangzhou Blood Center, and 24 blood samples from normal people were randomly selected in the experiment.

Main reagents: fetal bovine serum was purchased from GIBCO Company; RPMI1640 medium was purchased from INVERTROGEN Company; BHA was purchased from INVERTROGEN Company; anti-CD45RA-Texas Red, anti-CCR7-AF700\ anti-CD62L were detected by flow cytometry -PE-cy7 was purchased from BD Company; Lymphocyte Separation Solution was purchased from Tianjin Haoyang Biological Products Co., Ltd.; CD4 ⁺ T cell negative selection magnetic beads were purchased from BD Company.

Main experimental instruments: desktop high-speed centrifuge (Eppendorf Centrifuge 5810R), flow cytometer (Beckman Coulter), CO ₂ cell incubator (Thermo SCIENTIFIC), biological safety cabinet (ThermoSCIENTIFIC), inverted biological microscope (Leica), magnetic Bead sorting magnetic stand (BD Pharmigen).

cell culture

2.1 Cell extraction and separation

Mix the peripheral blood and PBS buffer (containing 2% BSA, 0.5% EDTA) at a ratio of 1:4; then slowly add the diluted blood to the top of the lymphocyte separation medium at a ratio of 1:1; centrifuge 300×g for 30 min; carefully absorb the mononuclear cells, put them into another centrifuge tube, add 5 times the volume of PBS (containing 0.5% BSA, 2% EDTA) to dilute, mix well, 300×g for 15 min; repeat the above One step at a time; incubate CD4+ T cell negative selection primary antibody for 15 min; dilute with 10 times the volume of PBS (containing 0.5% BSA, 2% EDTA), mix well, and incubate at 300×g for 12 min; incubate the secondary antibody with magnetic beads Antibody, 30 min; sort the cells through the column to obtain CD4+ T cells, add RPMI1640 containing 10% fetal bovine serum to resuspend the cells, count the cells and adjust the cells to the desired concentration.

2.2 Culture conditions

At 5% CO ₂ , saturated humidity and 37 ^o C, 5×10 ⁵ CD4 ⁺ T cells per well were cultured in 1 mL of RPMI1640 containing 10% fetal bovine serum.

Cell plating and drug treatment

3.1 planking

The sorted CD4 ⁺ T cells were plated in a 12-well plate. According to the experimental design, the expected number of cells per well was 5×10 ⁵ .

3.2 Dosing treatment

Five different treatments were performed on the plated CD4 ⁺ T cells, and three replicate wells were set up for each treatment as a parallel control, and corresponding stimulation was applied on the 4th day, 7th day, 10th day, and 13th day of cell culture .

Cell subgroup analysis and detection of cell number

After 15 days of cell culture, absorb the cells in the culture wells of different donors (n=24), and count them with a cell counter; absorb 5×10 ⁵ cells from the corresponding culture wells, 300×g for 15 min , Wash the cells twice with PBS, incubate the flow cytometry antibodies (CD45RA, CCR7, CD62L) for detecting CD4 ⁺ TCM for half an hour; wash off the flow cytometry antibodies, add PBS to dilute to the corresponding concentration, and use flow cytometry to detect. Both the control group and the experimental group were CD4 ⁺ T cells from the same donor, and cells from different donors were taken at each time point for detection.

Statistical software and statistical methods

SPSS13.0 analysis software was used for statistical analysis. All results of measurement data are expressed as mean ± standard deviation. The t test was used to compare the experimental group with the control group, and P<0.01 indicated a significant difference.

Experimental results

6.1 Flow cytometric detection of the proportion of CD4+TCM in CD4+T cells by BHA

In order to determine the initial ratio of TCM in CD4+ T cells stimulated by adding BHA, CD4 ⁺ T cells were spread in 24-well plates at 5×10 ⁶ cells/well, and anti-CD3 antibody, anti-CD28 antibody, IL-7 and IL-15. The cells in the control group and the experimental group were labeled with flow cytometry antibodies on day 15, and then the samples were detected by flow cytometry. From CD45RA-negative cells, CD62L and CCR7 double-positive cells were selected as the CD4 ⁺ TCM to be observed. According to the above-mentioned method of sorting CD4 ⁺ TCM by flow cytometry, the ratio of CD4 ⁺ TCM in CD4 ⁺ T cells was detected, as shown in FIG. 1 .

6.2 Detection of the effect of different concentrations of BHA on the expansion of CD4 ⁺ T cells

The time point on the 15th day of BHA treatment was selected for detection. The proportion of CD4 ⁺ TCM cells in the control group without BHA to the total CD4 ⁺ T cells was 16.06%; the proportion of BHA concentration was 5uM was 22.59%; the proportion of BHA concentration of 10uM 25.91%; BHA concentration of 20uM was 29.09%; BHA concentration of 40uM was 28.33%. The experimental results showed that the expansion of CD4 ⁺ T cells by different concentrations of BHA had a dose-dependent trend when it was lower than 20uM, as shown in Figure 2 .

Result analysis

For the first time, we used the small molecule drug BHA to promote the expansion of CD4 ⁺ TCM cells in vitro. Compared with the control group, the proportion of CD4 ⁺ TCM cells in the experimental group was significantly increased in CD4 ⁺ T cells.

In the adoptive immunotherapy of T cells, if a large number of adoptively transferred T cells will produce an overreaction, in the course of the experiment, we have found that a large number of initial T cells can be converted into CD4 ⁺ TCM cells under the condition that the total number of T cells remains unchanged. Methods. In the experiment, we further found out the optimal concentration of BHA that can promote the expansion of CD4 ⁺ TCM in vitro: 20uM. We found that after treating cells with a lower dose of BHA, there is a significant dose-dependent trend in the increase of the proportion of CD4+ TCM cells among CD4 ⁺ T cells; and when the concentration of BHA reaches 20uM, the effect of the increase in the proportion tends to be flat (Figure 2), this may be due to the excessive concentration of the drug, which greatly changes the growth environment of the cells and affects the normal growth of the cells. In this experiment, the cells were counted and detected after 15 days of BHA treatment because the initial number of CD4 ⁺ TCM in peripheral blood is very small, and it takes a long time for CD4 ⁺ T cells to synthesize in anti-CD3 and anti- More CD4 ⁺ TCMs were gradually formed under the co-stimulation of CD28. The experimental results also show that choosing to culture for a longer period of time before detection can obtain better results (Figure 2).

BHA is a commonly used food and drug antioxidant. In the present invention, the brand-new application of BHA in in vitro T cell culture is described for the first time, which provides good technical support for the extensive development of adoptive immunotherapy, and its molecular mechanism needs to be further studied in the future.

Claims (6)

1. the application that butylated hydroxy anisole is expanded in vitro to central memory t cell.

2. application according to claim 1, it is characterised in that the concentration of the butylated hydroxy anisole is 5 ~ 40 μM.

3. application according to claim 2, it is characterised in that the concentration of the butylated hydroxy anisole is 20 μM.

4. the application according to claims 1 to 3 any one, it is characterised in that the central memory t cell is CD4⁺ T cell.

5. application according to claim 1, it is characterised in that the extracting method of the central memory t cell is first will Periphery component blood presses 1 with the PBS containing 2% BSA and 0.5% EDTA:4 are well mixed；Then the blood after dilution is slowly added to The top of lymphocyte separation medium, the ratio of the two is 1:1；Centrifuge the min of 300 × g 30；It is careful to draw monocyte, insert In another centrifuge tube, the dilutions of the PBS containing 0.5% BSA and 2% EDTA of 5 times of volumes are added, after being well mixed, 300 × g are centrifuged 15 min；Repeat previous step once；It is incubated CD4⁺T cell the moon selects primary antibody, 15 min；Contain 0.5% BSA with 10 times of volumes Diluted with 2% EDTA PBS, after being well mixed, centrifuge the min of 300 × g 12；It is incubated the secondary antibody with magnetic bead, 30 min；Cross Post carries out cell sorting, obtains CD4⁺T cell.

6. the application that butylated hydroxy anisole according to claim 1 is expanded in vitro to central memory t cell, its It is characterised by, the cultural method of the central memory t cell is in 5% CO₂, under the conditions of saturated humidity and 37 DEG C by 5 × 10⁵ The CD4 in individual/hole⁺T cell is placed in RPMI1640 of 1 mL containing 10% hyclone and cultivated.