CN113416696B

CN113416696B - Immunoregulatory gamma delta T cell and in-vitro amplification method thereof

Info

Publication number: CN113416696B
Application number: CN202110710130.4A
Authority: CN
Inventors: 黄晓军; 刘江莹; 梁爽
Original assignee: Peking University Peoples Hospital
Current assignee: Peking University Peoples Hospital
Priority date: 2021-06-25
Filing date: 2021-06-25
Publication date: 2022-04-19
Anticipated expiration: 2041-06-25
Also published as: CN113416696A

Abstract

The invention discloses an immunoregulatory gamma delta T cell and an in-vitro amplification method thereof, wherein the immunoregulatory gamma delta T cell is a Reg-gamma delta T cell, and the only difference between the immunoregulatory gamma delta T cell and the gamma delta T cell is that the immunophenotype is CD3⁺Vδ2⁺CD25⁺CD127^‑. The method for expanding the Reg-gamma delta T cells is to adopt BMP2, zoledronic acid, IL-2 and IL-15 to induce healthy human PBMCs to generate a large amount of Reg-gamma delta T cells. The invention can not only amplify and obtain a large amount of Reg-gamma delta T cells, but also has short time (only 6-12 days), high proportion of gamma delta T cells and important application value.

Description

Immunoregulatory gamma delta T cell and in-vitro amplification method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an immunoregulatory gamma delta T cell and an in-vitro amplification method thereof, wherein the immunoregulatory gamma delta T cell is a Reg-gamma delta T cell, and the only difference between the immunoregulatory gamma delta T cell and the gamma delta T cell is that the immunophenotype is CD3⁺Vδ2⁺CD25⁺CD127^-。

Background

T lymphocytes are classified into α β T cells and γ δ T cells according to differences in human T cell antigen receptors (TCRs). In healthy adult peripheral blood, γ δ T cells account for CD3⁺1-20% of T cells have the characteristics of innate immunity and adaptive immunity, and play an important role in resisting infection and tumors.

The result of the mutual balance between the immune surveillance of the body and the immune escape of tumor cells determines the occurrence and development of tumors. Tumor cells can be induced to generate immunosuppressive cells by secreting inhibitory cytokines, or immunosuppressive factors are recruited by secreting chemokines, so that a tumor immunosuppressive environment is established, the activity and the anti-tumor effect of effector cells are inhibited, and the tumor progression is promoted. Accurate identification of regulatory immune cells in tumor environment and effective amplification are not only crucial to elucidating tumor immune escape mechanisms, but also provide potential therapeutic means for autoimmune diseases, chronic inflammatory diseases, graft-versus-host reactions and graft rejection reactions. The gamma delta T cell has the characteristic of directly recognizing antigen without depending on MHC molecules, and cannot cause allogenic reaction during foreign body feedback, so the gamma delta T cell has great application prospect in immunotherapy.

Due to the lack of effective amplification method of regulatory γ δ T cells, it is difficult to obtain sufficient number of cells for mouse model experiments and to meet the need of adoptive immunotherapy, and the generation mechanism and efficient culture and amplification scheme of regulatory γ δ T cells need further research.

Disclosure of Invention

The object of the present invention is how to perform immunotherapy. Further, an immunomodulatory cell (i.e., a Reg- γ δ T cell) is provided as an immunotherapy.

The invention firstly protects and amplifies a method for Reg-gamma delta T cells, which comprises the following steps:

(1) adding PBMCs, BMP2, zoledronic acid, IL-2 and IL-15 into the culture medium to obtain a culture system; then culturing for 2-4 days; the concentration of PBMCs in the culture system can be 3X 10⁶one/mL-5X 10⁶Per mL (e.g., 3X 10)⁶one/mL-4X 10⁶ 4X 10 units/mL⁶one/mL-5X 10⁶ 3X 10 pieces/mL⁶4X 10 units/mL⁶one/mL or 5X 10⁶BMP2 can be present in a concentration of 50-100ng/mL (e.g., 50-80ng/mL, 80-100ng/mL, 50ng/mL, 80ng/mL, or 100ng/mL), zoledronic acid can be present in a concentration of 1.5-3. mu. mol/mL (e.g., 1.5-2. mu. mol/mL, 2-3. mu. mol/mL, 1.5. mu. mol/mL, 2. mu. mol/mL, or 3. mu. mol/mL), IL-2 can be present in a concentration of 20-50ng/mL (e.g., 20-25ng/mL, 25-50ng/mL, 20ng/mL, 25ng/mL, or 50ng/mL), IL-15 can be present in a concentration of 40-60ng/mL (e.g., 40-50ng/mL, 50-60ng/mL, 40 ng/mL), 50ng/mL or 60 ng/mL);

(2) after the step (1) is completed, half of the culture solution is changed, and the culture is carried out for 2 to 4 days (such as 2 to 3 days, 3 to 4 days, 2 days, 3 days or 4 days);

(3) after the step (2) is completed, half of the culture solution is changed, and the culture is carried out for 2 to 4 days (such as 2 to 3 days, 3 to 4 days, 2 days, 3 days or 4 days); collecting Reg-gamma delta T cells in the system;

the only difference between Reg-gamma delta T cells and gamma delta T cells is that the immunophenotype of the former is CD3⁺Vδ2⁺CD25⁺CD127^-。

In the above method, in the step (1), the culture medium may have 8 to 12% (e.g., 8 to 10%, 10 to 12%, 8%, 10% or 12%) of fetal calf serum, 0.8 to 1.2% (e.g., 0.8 to 1.0%, 1.0 to 1.2%, 0.8%, 1.0% or 1.2%) of penicillin and 0.8 to 1.2% (e.g., 0.8 to 1.0%, 1.0 to 1.2%, 0.8%, 1.0% or 1.2%) of streptomycin, and the solvent is RPMI1640 complete culture medium (product of beijing fine engineering biotechnology limited, product No. AF 2953).

In the above method, in the step (2) and the step (3), the operation method of half-amount liquid change may be: half of the culture broth is removed, and half of the culture medium containing BMP2, IL-2, and IL-15 is added so that the concentration of BMP2 in the system can be 50-100ng/mL (e.g., 50-80ng/mL, 80-100ng/mL, 50ng/mL, 80ng/mL, or 100ng/mL), the concentration of IL-2 can be 20-50ng/mL (e.g., 20-25ng/mL, 25-50ng/mL, 20ng/mL, 25ng/mL, or 50ng/mL), and the concentration of IL-15 can be 40-60ng/mL (e.g., 40-50ng/mL, 50-60ng/mL, 40ng/mL, 50ng/mL, or 60 ng/mL). The culture medium may have 8-12% (such as 8-10%, 10-12%, 8%, 10% or 12%) of fetal calf serum, 0.8-1.2% (such as 0.8-1.0%, 1.0-1.2%, 0.8%, 1.0% or 1.2%) of penicillin and 0.8-1.2% (such as 0.8-1.0%, 1.0-1.2%, 0.8%, 1.0% or 1.2%) of streptomycin, and the solvent is RPMI1640 complete culture medium (Beijing Fine engineering Biotech Co., Ltd., product No. AF 2953).

The invention also protects Reg-gamma delta T cells; the only difference between Reg-gamma delta T cells and gamma delta T cells is that the immunophenotype of the former is CD3⁺Vδ2⁺CD25⁺CD127^-。

Reg-gamma delta T cells expanded by any of the methods described above are also within the scope of the invention.

The invention also protects a kit for amplifying the Reg-gamma delta T cells, which specifically comprises PBMCs, BMP2, zoledronic acid, IL-2 and IL-15; the only difference between Reg-gamma delta T cells and gamma delta T cells is that the immunophenotype of the former is CD3⁺Vδ2⁺CD25⁺CD127^-。

The invention also provides a kit for amplifying the Reg-gamma delta T cells, which consists of a culture medium, PBMCs, BMP2, zoledronic acid, IL-2 and IL-15; the only difference between Reg-gamma delta T cells and gamma delta T cells is that the immunophenotype of the former is CD3⁺Vδ2⁺CD25⁺CD127^-. The solute and its concentration of the culture medium can be 8-12% (such as 8-10%, 10-12%, 8%, 10% or 12%) of fetal calf serum, 0.8-1.2%% such as 0.8-1.0%, 1.0-1.2%, 0.8%, 1.0% or 1.2% penicillin and 0.8-1.2% (such as 0.8-1.0%, 1.0-1.2%, 0.8%, 1.0% or 1.2%) streptomycin in RPMI1640 complete medium (product of Beijing Fine engineering Biotech Co., Ltd., Cat. No. AF 2953).

The invention also protects the application of the Reg-gamma delta T cell, which can be at least one of A1) -A7);

A1) inhibiting PBMC cells;

A2) attenuating activation of CD4 cells and/or CD8 cells;

A3) reducing the secretion of inflammatory factors;

A4) inhibition of effector V δ 2 cells;

A5) reduction of the effective V.delta.2⁺Killer receptor expression of T cells;

A6) reduction of the effective V.delta.2⁺T cell inflammatory factor secretion;

A7) effectively inhibiting the anti-tumor effect of the effector V delta 2 cells;

The invention also protects the application of the Reg-gamma delta T cell, which can be at least one of B1) -B3);

B1) inhibition of effector T cells;

B2) immunotherapy or immunosuppression;

B3) as an immunomodulatory cell for immunotherapy;

In any of the above applications, the Reg- γ δ T cell may be a Reg- γ δ T cell expanded by any of the above methods.

The invention discovers a Reg-gamma delta T cell, which is different from the gamma delta T cell only in that: the immunophenotype of Reg-gamma delta T cells is CD3⁺Vδ2⁺CD25⁺CD127^-. The invention also establishes a method for in vitro cell proliferation by using various cytokines (BMP2, zoledronic acid,IL-2 and IL-15) induce healthy human PBMCs to produce large numbers of Reg-gamma delta T cells (immunophenotype is CD 3)⁺Vδ2⁺CD25⁺CD127^-) The method of (3) can amplify a large number of Reg-gamma delta T cells, has a short amplification time (only 6 to 12 days) and occupies a high proportion of gamma delta T cells. Reg- γ δ T cells have the following uses: suppression of PBMC cells, suppression of effector V delta 2 cells, suppression of effector T cells, as immunomodulatory cells for immunotherapy. The invention has important application value.

Drawings

FIG. 1 is a graph of the number of Reg- γ δ T cells in the AML tumor environment. a flow cytometry method for detecting CD3 in bone marrow blood⁺T cells, gamma delta T cells and V delta 2⁺T cell gating strategy; b and c are CD3 in bone marrow of healthy people and AML first-aid patients⁺CD25⁺CD127^-Vδ2⁺Flow detection of T cells (i.e., Reg-gamma delta T cells) typical images and statistical analysis results; d is the content of BMP2 in the bone marrow microenvironment of healthy people and AML initial patients; e is a correlation analysis of the proportion of Reg- γ δ T cells with BMP2 content in the AML bone marrow microenvironment.

FIG. 2 shows the in vitro expansion of Reg- γ δ T cells. a is the CD3 occupancy of gamma delta T cells after expansion in protocols 1-15⁺The ratio of T cells, the ratio of the Reg-gamma delta T cells to the gamma delta T cells after amplification and the absolute number change multiple of the Reg-gamma delta T cells before and after amplification are shown in the specification, wherein in the scheme 6, the concentration of BMP2 is 50ng/mL, the concentration of zoledronic acid is 2 mu mol/mL, the concentration of IL-2 is 25ng/mL, the ratio of the gamma delta T cells when the concentration of IL-15 is 50ng/mL, the ratio of the Reg-gamma delta T cells to the gamma delta T cells is higher, and the absolute number change multiple of the Reg-gamma delta T cells is the largest; b is a typical graph of Reg- γ δ T cell flow cytometry at day 0, day 3, day 6, and day 9 of protocol 6 amplification; and c is the statistical analysis of the proportion of the Reg-gamma delta T cells to the gamma delta T cells at the 0 th day, the 3 rd day, the 6 th day and the 9 th day of the PBMCs of different healthy people in the scheme 6, and the scheme 6 can be stably amplified in different healthy people.

FIG. 3 shows in vitro expanded Reg- γ δ T cells and effector V δ 2⁺Killing of AML cells by T cells (U937 cell line). a and b are that Reg-gamma delta T cells do not kill U937 by themselves, and the effectSex V delta 2⁺T cells can obviously kill U937 (P)<0.001) and the effective target ratio is 10: 1.

FIG. 4 is a graph of the inhibition of PBMC killing by Reg- γ δ T cells. a is that after the Reg-gamma delta T cells and PBMCs (removing the gamma delta T cells) are co-cultured, the killing effect of the PBMCs on U937 is weakened, and the weakening effect is gradually obvious along with the increase of the proportion of the Reg-gamma delta T cells; b-e shows that Reg-gamma delta T cells attenuate activation of CD4 cells (CD38, HLA-DR), reduce inflammatory factor secretion (IFN-gamma, TNF alpha); f-i showed that Reg- γ δ T cells attenuated activation of CD8 cells (CD38, HLA-DR), and reduced secretion of inflammatory factors (IFN- γ, TNF α).

FIG. 5 shows the response V.delta.2 to Reg-. gamma.delta.T cells⁺Inhibition of T cell killing. a is Reg-gamma delta T cell and effector V delta 2⁺After T cell co-culture, V.delta.2⁺The killing effect of the T cells on the U937 is weakened, and the weakening effect is gradually obvious along with the increase of the proportion of the Reg-gamma delta T cells; b-c shows that Reg-gamma delta T cells attenuate effector V delta 2⁺Activation of T cells (CD38, HLA-DR); d-e shows that Reg-gamma delta T cells reduce effector V delta 2⁺Killer receptor expression of T cells (DNAM1, NKG 2D); f-g shows that Reg-gamma delta T cells reduce effector V delta 2⁺T cell inflammatory factor secretion (IFN-. gamma., TNF. alpha.).

FIG. 6 shows the effect of expanded Reg-gamma delta T cells on effector V delta 2 in mice⁺Inhibition of T cells. a is a mouse grouping and pattern diagram; b is the imaging result of the mice in vivo on the 17 th day after the return transfusion; c-d are the results of flow detection of the proportion of U937 cells in peripheral circulation blood and bone marrow blood of the mice on day 17 respectively; e is the survival time statistics of different groups of mice.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples, healthy persons (21 total) and AML patients (21 total) gave informed consent to the experiments of the present invention.

The RPMI1640 complete culture medium is a product of Beijing Fine engineering Biotechnology Ltd, and the product number is AF 2953. Penicillin and streptomycin are both products of Beijing Fine engineering Biotechnology Ltd. 10 × hemolysin is a product of BD corporation, usa under the catalog number 349202. The Human BMP2 ELISA Kit is a product of Zhenzi Sichuan province, and the catalog number is HUFI 00041. Protein Transport Inhibitor was purchased from eBioscience under the cat number 00-4980-93. TCR gamma delta⁺celliosolatelationskit was purchased from Miltenyi Biotec under the accession number 130-. MACS buffer was purchased from Miltenyi Biotec, having a Cat number of 130-.

Example 1 Reg- γ δ T cell flow assay

The bone marrow blood to be tested is healthy human (n ═ 21) bone marrow blood or AML patient (n ═ 21) bone marrow blood.

1. Adding PE-Cy7-anti-human CD3, BV-510-anti-human-alpha beta T, PE-anti-human-V delta 2, FITC-anti-human-V delta 1, PE-Cy5-anti-human CD25 and BV-421-anti-human CD127 into 200 mul of bone marrow blood to be detected, mixing the bone marrow blood with light bomb, and incubating the mixture for 15min in a dark place;

2. after the step 1 is finished, adding 2ml of 1 Xhemolysin (formed by mixing 10 Xhemolysin and sterilized water for injection according to the volume ratio of 9: 1), flicking, uniformly mixing, and incubating for 8 min;

3. after step 2, centrifuging at 1300rpm for 5min, and collecting the precipitate;

4. after the step 3 is finished, taking the precipitate, adding 2ml of PBS buffer solution, centrifuging at 1500rpm for 5min, and collecting the precipitate;

5. after the step 4 is completed, taking the precipitate, adding 2ml of PBS buffer solution, centrifuging at 1500rpm for 5min, and collecting the precipitate;

6. after completion of step 5, the pellet was taken, resuspended in 200. mu.l PBS buffer and examined by flow cytometry.

The results are shown in FIGS. 1 a-c. The results show that the method has the advantages of high yield,CD3 in AML patient bone marrow compared to healthy persons⁺Vδ2⁺CD25⁺CD127^-The proportion of T cells (i.e., Reg-gamma delta T cells) was significantly increased.

Example 2 detection of factors in the bone marrow Environment of AML patients

The BMP2 antibody-coated 96-well plate, the washing solution, the BMP2 standard, the sample diluent, the biotinylated antibody working solution, the enzyme conjugate working solution, the color developing agent and the stop solution are all components in a Human BMP2 ELISA Kit.

The bone marrow blood to be tested is healthy human bone marrow blood (n ═ 21) or bone marrow blood of AML patients (n ═ 21).

1. Using a test tube without pyrogen and endotoxin, adding bone marrow blood to be tested, coagulating at room temperature for 30min, centrifuging at 1000g for 10min, and carefully separating serum.

2. The BMP2 antibody-coated 96-well plates were divided into 3 groups and treated as follows:

and (3) detection group: adding 100 μ l of serum into each well, sealing with sealing plate adhesive paper, and incubating at 37 deg.C for 90 min;

standard substance group: adding 100 μ l BMP2 standard with different concentrations into each well, sealing with sealing plate adhesive paper, and incubating at 37 deg.C for 90 min;

blank irradiation group: 100 μ l of sample dilution was added to each well.

3. After step 2 is completed, washing the plate for 4 times; the plate washing method comprises injecting 350 μ l of washing solution, and separating injection and aspiration for 15-30 s.

4. After

step

3, 100. mu.l of biotinylated antibody working solution was added to each well, the reaction wells were sealed with a sealing plate adhesive paper, and incubated at 37 ℃ for 60 min.

5. After the step 4 is finished, washing the plate for 4 times; the plate washing method comprises injecting 350 μ l of washing solution, and separating injection and aspiration for 15-30 s.

6. After completion of

step

5, 100. mu.l of the enzyme conjugate working solution was added to each well, the reaction wells were sealed with a sealing plate gummed paper, and incubated at 37 ℃ for 30 min.

7. After step 6, washing the plate for 4 times; the plate washing method comprises injecting 350 μ l of washing solution, and separating injection and aspiration for 15-30 s.

8. After completion of step 7, 100. mu.l of color developing agent was added to each well, protected from light, and incubated at 37 ℃ for 10-20 min.

9. After completion of

step

8, 100. mu.l of stop buffer was added to each well, and OD was detected immediately after mixing well_450nmThe value is obtained.

The results are shown in FIG. 1 as d-e. The results show that the proportion of Reg-gamma delta T cells in the bone marrow of AML patients is obviously related to the content of BMP2 in the bone marrow, and indicate that BMP2 in the bone marrow is probably an induction determinant of Reg-gamma delta T cells.

To further study the induction mechanism of Reg-gamma delta T cells and obtain a large number of Reg-gamma delta T cells in vitro, PBMCs from healthy humans were induced in vitro using BMP 2.

Example 3 in vitro expansion of Reg- γ δ T cells

First, preparation of human peripheral blood mononuclear cells (i.e., PBMCs)

1. Centrifuging peripheral blood of healthy people at 1300rpm for 5min, and collecting precipitate; then, the reaction solution was diluted with PBS buffer 1: diluting at a ratio of 1 to obtain a whole blood sample.

2. After step 1, taking a 15ml centrifuge tube, adding 4ml of human lymphocyte separation solution, and then carefully adding a fresh whole blood sample on the human lymphocyte separation solution very slowly along the tube wall; carefully put into a centrifuge and centrifuged at 2000rpm for 20 min.

The tube was carefully removed and observed. The uppermost layer is a pale red transparent plasma, the middle is a thin dense white annular layer (i.e., white membrane layer), then a centrifugal liquid layer, and the bottommost layer is a red blood cell layer.

3. After step 2, carefully absorbing the leucocyte layer by using a sterile plastic pipette, transferring into a new sterile conical centrifuge tube, adding 5 times volume of PBS buffer solution, uniformly mixing, centrifuging at room temperature and 2000rpm for 8min, discarding supernatant, re-suspending with 5ml of PBS, and centrifuging at room temperature and 2000rpm for 8 min.

4. After completion of step 3, the supernatant was discarded and resuspended in RPMI1640 medium containing 10% fetal calf serum, 1% penicillin and 1% streptomycin (the RPMI1640 medium containing 10% fetal calf serum, 1% penicillin and 1% streptomycin is hereinafter collectively referred to as 1640 complete medium) to obtain PBMCs.

Second, in vitro amplification of Reg-gamma delta T cells

1. Adding PBMCs obtained in step one into a 24-well plate containing 1mL of 1640 complete medium per well, and adjusting the density to 4X 10⁶/mL。

2. After the step 1 is completed, adding cytokines according to 15 schemes shown in table 1 to obtain a system (marked as day 0); the concentrations of the cytokines in the system are shown in Table 1.

TABLE 1 15 protocols for the induction of Reg- γ - δ T cells

Zoledronic acid

IL-15

IL-2

BMP2

IL-1β

activinA

Scheme

1

2μmol/mL

-

Scheme 2

2μmol/mL

50ng/mL

-

Scheme 3

2μmol/mL

50ng/mL

2ng/mL

50ng/mL

-

Scheme 4

2μmol/mL

50ng/mL

5ng/mL

50ng/mL

-

Scheme 5

2μmol/mL

50ng/mL

10ng/mL

50ng/mL

-

Scheme 6

2μmol/mL

50ng/mL

25ng/mL

50ng/mL

-

Scheme 7

2μmol/mL

50ng/mL

-

Scheme 8

2μmol/mL

50ng/mL

25ng/mL

5ng/mL

-

Scheme 9

2μmol/mL

50ng/mL

25ng/mL

10ng/mL

-

Scheme 10

2μmol/mL

50ng/mL

25ng/mL

100ng/mL

-

Scheme 11

2μmol/mL

50ng/mL

25ng/mL

-

5ng/mL

-

Scheme 12

2μmol/mL

50ng/mL

25ng/mL

-

10ng/mL

-

Scheme 13

2μmol/mL

50ng/mL

25ng/mL

-

30ng/mL

-

Scheme 14

2μmol/mL

50ng/mL

25ng/mL

-

10ng/mL

Scheme

15

2μmol/mL

50ng/mL

25ng/mL

-

50ng/mL

3. The system obtained in step 2 was cultured at 37 ℃ with 5% CO2 for 3 days (day 3).

4. After completion of step 3, the medium was changed half by half, and cultured at 37 ℃ with 5% CO2 for 3 days (day 6).

Cytokines other than zoledronic acid were added at half-change, and the concentration of all cytokines in the system is shown in table 1.

5. After completion of step 4, the medium was changed half by half, and cultured at 37 ℃ with 5% CO2 for 3 days (day 9).

6. The proportion of Reg-gamma delta T cells was measured by flow cytometry at day 0, day 3, day 6 and day 9, respectively.

The results of the partial detection are shown in FIG. 2.

The results show that the Reg-gamma delta T cells can be effectively amplified by the scheme 6, the scheme 7 and the scheme 10, wherein the proportion of the gamma delta T cells and the proportion of the Reg-gamma delta T cells in the gamma delta T cells are higher after the scheme 6 is amplified, the absolute number of the Reg-gamma delta T cells is increased to the maximum, the Reg-gamma delta T cells can be effectively amplified to the maximum, and the proportion of the Reg-gamma delta T cells is increased to 66.5% from 0.4% before induction. The result has better repeatability in different healthy people, proves that Reg-gamma delta T cells can be obtained in vitro in a large quantity stably by combining BMP2 with zoledronic acid, IL-2 and IL-15, and lays a good foundation for further verifying the functions and later transformation application of the cells.

Example 4 detection of killing Capacity of Reg-Gamma-Delta T cells

First, sorting Reg-gamma delta T cells

The sorting Biotin antibody mixed liquor and the anti-Biotin magnetic bead mixed liquor used for sorting are TCR gamma delta⁺Components in the celliosolatelationskit.

1. Take 1X 10⁷Example 3 Reg-. gamma.delta.T cells amplified in scheme 6, step two, were added to 4 mM ACS buffer, centrifuged at room temperature at 300g for 10min, and the supernatant carefully discarded.

2. After completion of

step

1, 80. mu.L of the resuspended cells were added and 20. mu.L of the Biotin antibody mixture was added and mixed and incubated at 4-8 ℃ for 10 minutes.

3. After completion of

step

2, 2 mM ACS buffer was added, centrifuged at 300g for 10min at room temperature, and the supernatant carefully discarded.

4. After completion of

step

3, 80. mu.L of the resuspended cells were added and mixed with 20. mu.L of the Lanti-Biotin magnetic bead mixture, and after mixing, the cells were incubated at 4 to 8 ℃ for 15 minutes.

5. After completion of

step

4, 2 mM ACS buffer was added, centrifuged at 300g for 10 minutes at room temperature, the supernatant carefully discarded, and resuspended in 500. mu.L of LMACS buffer for use.

6. The MS sorting column was placed on a MiniMACS sorter, and after 500. mu.LMACS buffer rinse, the cells obtained in step 5 were added and rinsed 3 times with 500. mu.LMACS buffer, and the flow-down cell suspension was collected.

7. After completion of step 6, centrifugation was carried out at 300g for 10 minutes at room temperature, the supernatant was carefully discarded, and the supernatant was resuspended in 1640 complete medium to obtain sorted Reg- γ δ T cells for use.

Second, detection of killing ability of sorted Reg-gamma delta T cells

1. U937 cells in logarithmic growth phase were taken and resuspended in 1640 complete medium, configured at 5X 10⁵200. mu.L of cell suspension was put on a 96 round bottom plate.

2. After completion of step 1, add 1X 10 per well⁶The selected Reg-gamma delta T cells in the step one are subjected to temperature regulation at 37 ℃ and 5% CO₂And culturing for 4 h.

3. After completion of step 2, the cells were centrifuged, collected and washed 2 times (each wash was made by adding 2mL of PBS buffer for resuspension, centrifuging at 1500rpm for 5min, and discarding the supernatant).

4. After the step 3 is completed, labeling FITC-anti-human-V delta 2, flicking and mixing evenly, and incubating for 15min in a dark place.

5. After completion of step 4, 2mL of PBS buffer was added for resuspension, centrifuged at 1500rpm for 5min, and the supernatant was discarded.

6. After completion of

step

5, 100. mu.L of PBS buffer was added to resuspend the cells, 7AAD was added, gently mixed, and incubated for 15min in the dark.

7. After completion of step 6, 100. mu.L of PBS buffer was added and detected by flow cytometry within 1 h.

The results are shown in FIG. 3. The results indicate that sorted Reg- γ δ T cells do not have the ability to kill U937 cells.

Example 5 inhibition of PBMC cell Effect by Reg- γ δ T cells

Each group was treated as follows:

1. the method comprises the following five groups:

taking 5X 10⁴U937 cells, 37 ℃, 5% CO₂And culturing for 24 h.

② 5 × 10⁴U937 cells and 50X 10⁴PBMCs (PBMCs) depleted of gamma delta T cells_Δγδ) Mixing at 37 deg.C with 5% CO₂Culturing for 20h, taking out half of the cells, adding protein transport Inhibitor, recording the concentration of 0.2 μ L/100 μ L as template 1, recording the rest cells as template 2, 37 deg.C, and 5% CO₂The cultivation was continued for 4 h.

③ 5 is multiplied by 10⁴U937 cells, 50X 10⁴PBMC (peripheral blood mononuclear cell)_ΔγδAnd 2.5X 10⁴The sorted Reg-gamma delta T cells of step one of example 4 were mixed at 37 ℃ with 5% CO₂Culturing for 20h, taking out half of the cells, adding Protein Transport Inhibitor, recording the concentration of 0.2 μ L/100 μ L as template 1, recording the rest cells as template 2, 37 deg.C, and 5% CO₂The cultivation was continued for 4 h.

Fourthly, mixing 5 multiplied by 10⁴U937 cells, 50X 10⁴PBMC (peripheral blood mononuclear cell)_ΔγδAnd 5X 10⁴The sorted Reg-gamma delta T cells of step one of example 4 were mixed at 37 ℃ with 5% CO₂Culturing for 20h, taking out half of the cells, adding Protein Transport Inhibitor, recording the concentration of 0.2 μ L/100 μ L as template 1, recording the rest cells as template 2, 37 deg.C, and 5% CO₂The cultivation was continued for 4 h.

Fifthly, 5 is multiplied by 10⁴U937 cells, 50X 10⁴PBMC (peripheral blood mononuclear cell)_ΔγδAnd 10X 10⁴The sorted Reg-gamma delta T cells of step one of example 4 were mixed at 37 ℃ with 5% CO₂Culturing for 20h, taking out half of the cells, adding Protein Transport Inhibitor, recording the concentration of 0.2 μ L/100 μ L as template 1, recording the rest cells as template 2, 37 deg.C, and 5% CO₂The cultivation was continued for 4 h.

2. After completion of step 1, the cells of template 1 were centrifuged, collected and washed 2 times (each wash was made by adding 2mL of PBS buffer for resuspension, centrifuging at 1500rpm for 5min, and discarding the supernatant).

3. After step 2 is completed, labeling cells of the template 1 with APC-anti-human-CD8, BV605-anti-human-CD4, APC-Cy7-anti-human-HLA-DR and PE-anti-human-CD38, flicking, mixing uniformly, and incubating for 15min in the dark.

4. After completion of step 3, 2mL of PBS buffer was added to the cells of template 1 for resuspension, centrifuged at 1500rpm for 5min, and the supernatant was discarded.

5. After completion of

step

4, 100. mu.L of fixative/rupture agent A was added to the cells of template 1 and incubated for 15min at room temperature in the dark.

6. After completion of step 5, 2mL of PBS buffer was added to the template 1 cells for resuspension, centrifuged at 1500rpm for 5min, and the supernatant was discarded.

7. After completion of step 6, 100. mu.L of fixation/membrane-rupture agent B, BV421-anti-human-IFN γ and PE-anti-human-TNF- α were added to the cells of template 1 and incubated for 15min at room temperature in the absence of light.

8. After completion of step 7, 2mL of PBS buffer was added to the cells of template 1 for resuspension, centrifuged at 1500rpm for 5min, and the supernatant was discarded.

9. After completion of

step

8, 200. mu.L of PBS buffer was added to the cells of template 1 and detected by flow cytometry.

10. The cells of template 2 were centrifuged, the cells were collected and washed 2 times (each wash was performed by adding 2mL of PBS buffer for resuspension, centrifuging at 1500rpm for 5min, and discarding the supernatant).

11. After the step 10 is completed, the cell marker PE-cy7-anti-human-CD3 of the template 2 is flicked and mixed evenly, and incubated for 15min in the dark.

12. After completion of step 11, 2mL of PBS buffer was added to the cells of template 2 for resuspension, and centrifuged at 1500rpm for 5min, and the supernatant was discarded.

13. After completion of step 12, 100 μ L of PBS buffer was added to the cells of template 2 to resuspend the cells, 7AAD was added, gently mixed, and incubated for 15min in the dark.

14. After completion of step 13, 100. mu.L of PBS buffer was added and detected by flow cytometry within 1 h.

The results are shown in FIG. 4. The result shows that after the Reg-gamma delta T cells and the PBMCs are co-cultured, the killing effect of the PBMCs on the U937 is weakened, and the weakening effect is gradually obvious along with the increase of the proportion of the Reg-gamma delta T cells; the Reg-gamma delta T cells weaken the activation of CD4 cells and reduce the secretion of inflammatory factors; reg-gamma delta T cells attenuate activation of CD8 cells and reduce secretion of inflammatory factors.

Example 6 Reg- γ δ T cells inhibit effector V δ 2 cell effects

One, responsive V delta 2 cell preparation

1. A24-well plate containing 1mL of 1640 complete medium per well was taken, and PBMCs obtained in the first step of example 3 were added thereto at a density of 2X 10⁶/mL。

2. After completion of step 1, on day 1, pamidronate was added to a concentration of 9. mu.g/mL in the system, and the system was charged with 5% CO at 37 ℃₂Culturing in an incubator.

3. After completion of step 2, the experiment was performed by changing the medium on day 4, centrifuging at 1500rpm for 5min, removing the supernatant, adding 1640 complete medium, supplementing pamidronate to a concentration of 9. mu.g/mL in the system, supplementing recombinant human IL-2 to a concentration of 50ng/mL in the system, 37 ℃, 5% CO₂And (5) culturing.

4. After completion of step 3, the experiment was performed by changing the medium on day 7, centrifuging at 1500rpm for 5min, removing the supernatant, adding 1640 complete medium, supplementing recombinant human IL-2 to a concentration of 50ng/mL in the system, 37 ℃ and 5% CO₂And (5) culturing.

5. After completion of step 4, the experiment was performed by changing the medium on day 9, centrifuging at 1500rpm for 5min, removing the supernatant, adding 1640 complete medium, supplementing recombinant human IL-2 to a concentration of 50ng/mL in the system, 37 ℃ and 5% CO₂And (5) culturing.

6. After completion of step 5, the culture was changed on day 11, centrifuged at 1500rpm for 5min, the supernatant was removed, and 1640 complete medium was added to supplement recombinant human IL-2 at a concentration of 50ng/mL in the system, 37 ℃ and 5% CO₂And (5) culturing.

7. After completing step 6, culturing for 12-14 days to obtain culture purity>90% of effector V delta 2 cells, centrifuging at 1500rpm for 5min, removing supernatant, adding calcein-AM working solution (concentration of 2.5 μ M) at 37 deg.C and 5% CO₂Culturing in the environmentIncubate with light for 30 minutes.

8. After completion of step 7, the cells were washed 2 times (each washing was performed by adding 2mL of 1640 complete medium, centrifuging at 1500rpm for 5min, and discarding the supernatant).

9. After completion of step 8, 1mL of complete medium was added to obtain effector V δ 2 cells for use.

Two, Reg-gamma delta T cell inhibition effector V delta 2⁺T cells

Recovering the sorted Reg-gamma delta T cells and the effector V delta 2 in step one of example 4⁺T cells were grouped and processed as follows:

1. the method comprises the following five groups:

taking 5X 10⁴U937 cells, 37 ℃, 5% CO₂And culturing for 6 h.

② 5 × 10⁴U937 cells and 50X 10⁴Effective V delta 2⁺T cell mix, 5% CO at 37 ℃₂After 6h of culture, the plate 2 was recorded, and the Protein Transport Inhibitor was added to the other well under the same conditions, and 0.2. mu.L/100. mu.L of the plate 1 was recorded.

③ 5 is multiplied by 10⁴U937 cells, 50X 10⁴Effective V delta 2⁺T cells and 2.5X 10⁴Mixing the cells with Reg-gamma-delta T cells at 37 deg.C and 5% CO₂After 6h of culture, the plate 2 was recorded, and the Protein Transport Inhibitor was added to the other well under the same conditions, and 0.2. mu.L/100. mu.L of the plate 1 was recorded.

Fourthly, mixing 5 multiplied by 10⁴U937 cells, 50X 10⁴Effective V delta 2⁺T cells and 5X 10⁴Mixing the cells with Reg-gamma-delta T cells at 37 deg.C and 5% CO₂After 6h of culture, the plate 2 was recorded, and the Protein Transport Inhibitor was added to the other well under the same conditions, and 0.2. mu.L/100. mu.L of the plate 1 was recorded.

Fifthly, 5 is multiplied by 10⁴U937 cells, 50X 10⁴Effective V delta 2⁺T cells and 10X 10⁴Mixing the cells with Reg-gamma-delta T cells at 37 deg.C and 5% CO₂After 6h of culture, the plate 2 was recorded, and the Protein Transport Inhibitor was added to the other well under the same conditions, and 0.2. mu.L/100. mu.L of the plate 1 was recorded.

3. After step 2 is completed, cell markers PE-Cy7-anti-human-CD3, APC-Cy7-anti-human-HLA-DR, PE-anti-human-CD38, APC-Cy7-anti-human-NKG2D and BV605-anti-human-DNAM1 of the template 1 are flicked and mixed evenly, and incubated for 15min in a dark place.

5. After completion of

step

6. After completion of step 5, 2mL of PBS buffer was added to the cells of template 1 for resuspension, centrifuged at 1500rpm for 5min, and the supernatant was discarded.

9. After completion of

step

The results are shown in FIG. 5. The results show that Reg-gamma delta T cells and effector V delta 2⁺After T cell co-culture, V.delta.2⁺The killing effect of the T cells on the U937 is weakened, and the weakening effect is gradually obvious along with the increase of the proportion of the Reg-gamma delta T cells; reg-gamma delta T cell attenuation of effector V delta 2⁺Activation of T cells; reg-gamma delta T cell reduction of effector V delta 2⁺Killer receptor expression of T cells; reg-gamma delta T cell reduction of effector V delta 2⁺T cell inflammatory factors are secreted.

Example 7 Reg- γ δ T cell inhibition in mouse model

NOD-Prkdc of severely immunodeficient mice^scidIL2rg^tm1the/Vst (NPG mouse for short) is a product of Beijing Wintoda laboratory animal technology company Limited.

6-8 week-old female NPG mice were randomly divided into four groups, 5 each, of PBS group (negative control), U937 group (U937 cells only reinfused, positive control group), V delta 2 group (U937 cells reinfused and effector V delta 2 cells) and Reg-gamma delta T group (U937 cells reinfused, effector V delta 2 cells and Reg-gamma delta T cells sorted in step one of example 4), and treated as follows:

PBS group (negative control): on the 0 th, 1 st and 3 rd days of the experiment, PBS buffer solution is injected into tail vein; on day 17 of the experiment, mice were bled orbitally and small animals were imaged whole body in vivo.

Group U937: day 0 of the experiment, tail vein injection 1X 10⁵U937 cells transfected with Luciferase (Luciferase); on

days

1 and 3 of the experiment, the tail vein was injected with PBS buffer; on day 17 of the experiment, mice were bled orbitally and small animals were imaged whole body in vivo.

Group V δ 2: day 0 of the experiment, tail vein injection 1X 10⁵U937 cells transfected with Luciferase (Luciferase); experiment day 1 and day 3, tail vein injection 2X 10⁷ Effector V delta 2 cells; on day 17 of the experiment, mice were bled orbitally and small animals were imaged whole body in vivo.

Reg- γ δ T group: day 0 of the experiment, tail vein injection 1X 10⁵U937 cells transfected with Luciferase (Luciferase); experiment day 1 and day 32X 10 for tail vein injection respectively⁷ Effective V delta 2 cells and 4X 10 cells⁶Reg-gamma delta T cells; on day 17 of the experiment, mice were bled orbitally and small animals were imaged whole body in vivo.

During the experiment, the mobility of the mice is observed until the mice die, and the hind limb femoral bone marrow of the mice is taken for flow cytometry detection.

The results of the experiment are shown in FIG. 6. In vivo fluorescence imaging of mice on day 17 after cell reinfusion revealed that the AML tumor burden in mice was greater in the U937 group than in the Reg- γ δ T group, and significantly greater in the Reg- γ δ T group than in the V δ 2 group; detecting peripheral circulating blood of the mice on the 17 th day to find that the proportion of the U937 cells in each group is consistent with the living body imaging result; within 30 minutes of the spontaneous death of the mice, the proportion of AML cells in the bone marrow of the mice is also greater in the U937 group than in the Reg-gamma delta T group, and greater in the latter than in the V delta 2 group; statistics of 32-day death conditions of the mice shows that the average survival time of the U937 group is shortest, the average survival time of the V delta 2 group is longest, and the survival of the vs U937 group of the V delta 2 group and the survival of the vsV delta 2 group of the Reg-gamma delta T group are obviously different. The results prove that the Reg-gamma delta T cells can effectively inhibit the antitumor effect of the effector V delta 2 cells in a mouse body and have the in-vivo immunoregulation treatment potential.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims

1. A method of expanding Reg- γ δ T cells, comprising the steps of:

(1) adding PBMCs, BMP2, zoledronic acid, IL-2 and IL-15 into the culture medium to obtain a culture system; then cultureCulturing for 2-4 days; the concentration of PBMCs in the culture system was 3X 10⁶one/mL-5X 10⁶BMP2 at a concentration of 50-100ng/mL, zoledronic acid at a concentration of 1.5-3 μmol/mL, IL-2 at a concentration of 20-50ng/mL, and IL-15 at a concentration of 40-60 ng/mL;

(2) after the step (1) is finished, changing the liquid in half, and culturing for 2-4 days;

(3) after the step (2) is finished, changing the liquid in half, and culturing for 2-4 days; collecting Reg-gamma delta T cells in the system;

2. The method of claim 1, wherein: in the step (1), the solute and the concentration of the culture medium are 8-12% of fetal calf serum, 0.8-1.2% of penicillin and 0.8-1.2% of streptomycin, and the solvent is RPMI1640 complete culture medium.

3. The method of claim 1, wherein: in the step (2) and the step (3), the operation method for half-amount liquid change comprises the following steps: half of the culture solution is removed, and half of the culture medium containing BMP2, IL-2 and IL-15 is added to make the concentration of BMP2 in the system be 50-100ng/mL, the concentration of IL-2 be 20-50ng/mL and the concentration of IL-15 be 40-60 ng/mL.

4. The method of claim 3, wherein: the solute and its concentration of the culture medium are 8-12% fetal calf serum, 0.8-1.2% penicillin and 0.8-1.2% streptomycin, and the solvent is RPMI1640 complete culture medium.

5. The method of claim 1, wherein: the culture time is 2-4 days, namely 3 days.

A Reg- γ δ T cell, characterized by: prepared by the method of any one of claims 1 to 5; the only difference between the Reg-gamma delta T cells and the gamma delta T cells is that the immunophenotype of the Reg-gamma delta T cells is CD3⁺Vδ2⁺CD25⁺CD127^-。

7. The kit for amplifying the Reg-gamma delta T cells is a kit A or a kit B; the only difference between Reg-gamma delta T cells and gamma delta T cells is that the immunophenotype of the former is CD3⁺Vδ2⁺CD25⁺CD127^-；

The kit A consists of PBMCs, BMP2, zoledronic acid, IL-2 and IL-15;

the kit B consists of a culture medium, PBMCs, BMP2, zoledronic acid, IL-2 and IL-15;

the solute and its concentration of the culture medium are 8-12% fetal calf serum, 0.8-1.2% penicillin and 0.8-1.2% streptomycin, and the solvent is RPMI1640 complete culture medium.

8. The use of the Reg- γ δ T-cell of claim 6, which is at least one of A1) -A7);

A1) inhibiting PBMC cells;

A2) attenuating activation of CD4 cells and/or CD8 cells;

A3) reducing the secretion of inflammatory factors;

A4) inhibiting the effective V delta 2⁺A T cell;

A7) effective inhibition of effective V delta 2⁺Anti-tumor effects of T cells;

the use is for the diagnosis and treatment of non-diseases.

9. Use of a Reg- γ δ T-cell according to claim 6 for inhibiting an effector T-cell;

the use is for the diagnosis and treatment of non-diseases.