CN113881631B

CN113881631B - Tonsil-derived T γδ Cell and preparation method and application thereof

Info

Publication number: CN113881631B
Application number: CN202111182222.6A
Authority: CN
Inventors: 张欢; 于洋; 程铧
Original assignee: Beijing Yibo Puhui Biotechnology Development Co ltd
Current assignee: Beijing Yibo Puhui Biotechnology Development Co ltd
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2022-08-02
Anticipated expiration: 2041-10-11
Also published as: CN113881631A

Abstract

The application relates to T _γδ The technical field of cell preparation, in particular to a tonsil-derived T _γδ Cells, methods of making and uses thereof. T is _γδ A method of making a cell comprising the steps of: s1, separating immune cells from the tonsil; s2, contacting immune cells with Tax protein of STLV4 virus, and preparing dendritic cells after cell culture; s3, mixing immune cell prepared in step S1 with dendritic cell, and culturing to obtain T in cell culture _γδ A cell. T of the present application _γδ The cells can be used for preparing anti-tumor or anti-virus cell products; t of the present application _γδ After 3 weeks of in vitro culture, the cells still had excellent antitumor effect.

Description

Tonsil-derived T γδ Cell and preparation method and application thereof

Technical Field

The application relates to T _γδ The technical field of cell preparation, in particular to a tonsil-derived T _γδ Cells, methods of making and uses thereof.

Background

Tonsils are immunologically active organs that are the first line of defense of the immune system against ingested or inhaled foreign pathogens, and are therefore often engorged with blood to help mount an immune response to common diseases such as respiratory viral infections. However, repeated tonsillitis seriously affects the quality of life, so tonsillectomy becomes the most commonly performed operation in the department of otorhinolaryngology to treat repeated chronic tonsillitis. It is known that the mucosa of tonsil contains a large amount of lymphoid tissue and is the place where various immune cells are accumulated. The immune cells in the removed tonsil are fully utilized, cultured and activated, and then can be used for treating autologous or allogeneic cancers or virus infectious diseases, and can also be used for reconstruction treatment after immune function imbalance.

The amygdala contains resident dendritic cells, a special antigen presenting cell, which can take up antigens produced by pathogens. Such antigen-loaded dendritic cells can then further activate the underlying T and B lymphocytes in the tonsils, thereby generating an immune response to the pathogen. T cells can be divided into two classes according to the difference in T Cell Receptors (TCR): t is _αβ Cells and T _γδ A cell. Known as T _γδ The cell is a T cell which can play a role of resisting pathogens or cancer cells without depending on MHC molecules, can be applied to autologous or allogeneic T with high activity _γδ The cells have great clinical application value.

At present, T _γδ The cells are conventionally prepared by collecting mononuclear cells (PBMCs) in peripheral blood, and culturing them with IL2 and phosphate small molecule compounds (e.g., zoledronic acid, etc.). However, it has been reported that this method has a short amplification cycle (generally about 14 days, the amplification amount is limited), and T is obtained _γδ The survival time of the cells is short (the expanded cells can continue to survive for about 7 days), the anti-apoptosis ability of the cells is poor, and the killing ability to tumor cells is poor.

Disclosure of Invention

In order to further improve the T obtained by preparation _γδ Tumor cell killing ability of cells and prolongation of T _γδ Survival time of cells, the present application provides a tonsil-derived T _γδ Cells, methods of making and uses thereof.

In a first aspect, the present application provides a tonsil derived T _γδ The preparation method of the cell adopts the following technical scheme:

tonsil-derived T _γδ A method of making a cell comprising the steps of:

s1, separating immune cells from the tonsil;

s2, taking immune cells to contact with Tax protein of the STLV4 virus, and preparing dendritic cells after cell culture;

s3, mixing immune cell prepared in step S1 with dendritic cell, and culturing to obtain T in cell culture _γδ A cell.

By adopting the technical scheme, the immune cells are separated from the tonsil, the Tax protein of the STLV4 virus is selected to contact with the prepared immune cells, and most of the dendritic cells (namely DC cells) colonized by the tissue prepared by the method are CD141+ subtype. Selecting the DC cell to prepare T _γδ When the cell is cultured, the cell is tested,the T is _γδ After 3 weeks of in vitro cell culture, the cells still have strong antitumor effect.

Preferably, the gene sequence for coding the Tax protein is shown as SEQ ID NO.1, and the amino acid sequence of the Tax protein is shown as SEQ ID NO. 2.

Preferably, the method for preparing the Tax protein of the STLV4 virus comprises the steps of inserting the coding gene of the Tax protein of the STLV4 virus into a lentiviral vector, transfecting a mixed packaging plasmid into a host cell, culturing the host cell and isolating the Tax protein of the STLV4 virus.

Preferably, in step S2, 5 to 15 mug/mL of dextran is further added when the Tax protein of the STLV4 virus is contacted with the immune cells.

By adopting the technical scheme, the efficiency of infecting immune cells by the Tax protein of the STLV4 virus can be effectively improved.

Preferably, in step S2, in the cell culture, 30-80 unit/mL of IL2 is further added, and 30-80 unit/mL of IL2 is added after the culture medium is replaced every 2-4 days.

By adopting the technical scheme, the high-dose IL2 tends to induce T cell expansion, and in the process of preparing DC cells, the T cells can occupy nutrients in a culture medium after being proliferated too fast, so that the growth of the DC cells is slowed down, and the DC cells are finally failed to be prepared. Therefore, the IL2 with the dosage can effectively promote the growth of DC cells, and is beneficial to obtaining more DC cells. In addition, the new culture medium is replaced every 2-4 days, and 30-80 unit/mL of IL2 is added, so that the effect of delaying the growth of T cells is achieved.

Preferably, the step of separating immune cells from tonsil in step S1 includes the steps of cutting tonsil into pieces, sieving with a 175-225 mesh sieve under the action of external force to obtain dispersed cells, and centrifuging the dispersed cells with Ficoll-Hypaque solution.

Preferably, the Ficoll-Hypaque centrifugation comprises the following steps:

I. placing the dispersed cells on the Ficoll-Hypaque liquid surface lightly and centrifuging under the condition of 800-1000 g;

II. After collecting leucocyte, supplementing physiological saline and centrifuging under the condition of 800-1000 g; and (4) discarding the supernatant, supplementing physiological saline again, and centrifuging under the condition of 800-1000 g.

Preferably, during the centrifugation in the step I, the speed increasing is 3, and the speed decreasing is 0; during centrifugation in step II, the acceleration is 9 and the deceleration is 6.

Preferably, 80% -95% of the dendritic cells prepared in step S2 are CD141+ subtypes.

By adopting the technical scheme, CD141+ cell subtype is reported to have stronger effect of cross presenting soluble antigen, and the cell of the subtype is rare in blood species but is considered to be the most important DC cell for activating CD8+ T cells to play the role of anti-tumor and anti-virus.

In a second aspect, the present application provides a tonsil derived T _γδ The cell adopts the following technical scheme:

tonsil-derived T _γδ Cells of the T _γδ The cells are prepared by the preparation method.

Preferably, said T is _γδ Cell-expressed markers include markers that are commonly found in antigen presenting cells such as CD80 and CD86, the NK cell activation receptor NKG2D, and the cytotoxic T cell marker TRAIL.

In a third aspect, the present application provides a tonsil derived T _γδ The application of the cell in preparing the cell product for resisting tumor or virus adopts the following technical scheme:

tonsil-derived T _γδ Use of cells for the preparation of an antitumor or antiviral cell product, said T _γδ The cells are prepared by the preparation method.

In summary, the present application has the following beneficial effects:

1. the tonsil contains a large amount of immune cells, the tonsil is fully utilized, dendritic cells (namely DC cells) are prepared from tonsil-derived immune cells, the immune cells prepared by the method are derived from a medullary line (CD 11c +) and show maturation (CD 83 +) and activation phenotype (HLA-DR +/CCR7+/CD80+/CD86 +), and in addition, most cells show CD1The 41+ subtype; then co-culturing the DC cell and the immune cell to finally prepare the T _γδ After 3 weeks of in vitro culture, the cells still had excellent antitumor effect.

2. In the present application, when the immune cells are contacted with the Tax protein of the STLV4 virus and then cultured, it is preferable to promote proliferation of DC cells by using IL2 of 30-80 unit/mL to obtain more DC cells.

3. Will be T of the present application _γδ When the cells are used for resisting tumors or viruses, the cells have excellent antitumor or viral effects.

Drawings

FIG. 1 is the results of flow cytometric analysis of DC cells according to an embodiment of the present application;

FIG. 2 is an illustration of DC cell-mediated T in tonsils according to an embodiment of the present application _γδ Flow cytometric analysis of the cells;

FIG. 3 is T of example 1 _γδ The result of the cell acting on the cervical cancer cell HeLa; wherein FIG. 3-1 is a view showing a result of microscopic observation, and FIG. 3-2 is a view showing a result at T _γδ Cell survival rate of cervical cancer cell HeLa under cell action.

FIG. 4 shows T in example 1 _γδ Results of cellular effects on melanoma cells a 375; wherein FIG. 4-1 is a view showing the result of microscopic observation, and FIG. 4-2 is a view showing the result at T _γδ Cell survival of melanoma cell a375 by cell action.

Detailed Description

The present application will be described in further detail with reference to the drawings and examples.

Examples of preparation of lentivirus

The lentivirus plasmid vector construction and lentivirus preparation method comprises the following steps:

(1) inserting a gene fragment of a Tax protein of the STLV4 virus into a lentiviral vector, transforming the recombinant plasmid vector into E.coli, sequencing correctly, shaking bacteria, and extracting the recombinant plasmid vector for later use.

(2) Mixing the obtained lentiviral plasmid vector of the STLV4 virus with a mixed packaging plasmid (the mixed packaging plasmid comprises expression plasmids of VSV-G, Gag-Pol and Rev), and adding a transfection reagent; the transfection reagent may be a calcium phosphate transfection reagent, a lipofection reagent, or a high molecular weight polymer transfection reagent. The lentiviral plasmid vector of the STLV4 virus was then co-transfected with the mixed packaging plasmid into host cells, which may be 293 cells, 293T cells or 293FT cells, for co-culture.

(3) Concentrating

The concentration method may be the following method 1 or method 2, and may further be:

the method comprises the following steps: collecting virus supernatant, and centrifuging at 23000-28000 rpm at 0-4 deg.C for 1.5-2.5 h.

The method 2 comprises the following steps: and adding PEG8000 and NaCl solution with proper concentration into the virus supernatant obtained after co-culture, and centrifuging for 30-60 min at the rotation speed of 1400-1800 g or 2500-3500 rpm under the condition of normal temperature or 0-4 ℃.

(4) And then, abandoning the supernatant, resuspending the obtained virus precipitate by using an RPMI1640 culture medium, and freezing and storing.

In one embodiment, the lentiviral plasmid vector construction and lentiviral vector preparation method comprises the steps of:

(1) the gene sequence of the Tax protein of the STLV4 virus is shown as SEQ ID number 1, a Jinwei company is entrusted to perform codon optimization and then synthesize, a lentiviral vector EcoRI-BamHI site is inserted, the recombinant plasmid vector is converted into E.coli, after sequencing is correct, bacteria are shaken, and a plasmid is extracted by a plasmid extraction kit for later use.

(2) The obtained lentiviral plasmid vector of STLV4 virus was mixed with a mixed packaging plasmid (available from Invitrogen, including expression plasmids for VSV-G, Gag-Pol and Rev) and after the addition of a polyfect reagent (available from Qiagen), 293 cells (available from ATCC) were co-transfected.

(3) Concentration: the virus supernatant was collected, precipitated with PEG8000 overnight, and centrifuged the next day at 1600g for 1h at 4 ℃.

(4) The supernatant was discarded, and the resulting viral pellet (i.e., STLV4 Tax lentivirus) was resuspended in RPMI1640 medium and frozen at-80 ℃ in a freezer.

Examples

Example 1

Tonsil-derived T _γδ A method of making a cell comprising the steps of:

s1 separating immune cells from tonsil

A method of isolating immune cells from tonsils, comprising the steps of:

s11, sample transportation and pretreatment: extracting fresh tonsils from individuals subjected to conventional tonsillectomy, putting the fresh tonsils into RPMI1640 culture medium containing streptomycin, transporting to a laboratory under the condition of a cold chain at 2-8 ℃, and completing extraction within 1-3 h.

In a biosafety cabinet, using sterile forceps, a sample of fresh tonsils was placed on a sterile 10cm first cell culture dish and tissue was kept moist with 2mL of HANK' S buffer containing antibiotics; then, a specimen of the fresh tonsil is cut into small tissue blocks of 3-5 mm by using scissors.

S12, preparing immune cell suspension:

s121, placing the sterilized stainless steel screen mesh (200 meshes) into a sterile 10cm second cell culture dish;

s122, adding 2mL of HANK' S buffer solution into the sterile second cell culture dish; placing a small tissue block on a stainless steel screen by using a sterile forceps; squeezing the tissue pieces using the plunger portion of a 5mL plastic syringe to push lymphocytes present in the tissue pieces through the screen; then washing the rest tissues with HANK' S buffer solution for two to three times until the separated cells on the tissues are washed clean, and finally obtaining a cell suspension on a second cell culture dish; all cell suspensions were transferred from the second cell culture dish to a 50mL centrifuge tube with a pipette.

And S123, repeating the step S122 to elute the immune cells in the remaining tissue small block in the first cell culture dish.

S13 centrifugation

The cell suspension after elution was transferred to a sterile centrifuge tube and the cells were repeatedly gently blown to disperse the cell pellet.

Then, 15mL of Ficoll-Hypaque solution is added into a clean 50mL centrifugal tube, the cell suspension is gently added on the surface of the Ficoll-Hypaque solution, and then the solution is centrifuged for 20min at the room temperature of 1000g, the rising speed is 3, and the falling speed is 0; collecting leucocyte, supplementing to 40mL with physiological saline, and centrifuging at 1000g and room temperature for 10min at an ascending rate of 9 and a descending rate of 6; the supernatant was discarded, the cells were resuspended in 40mL of physiological saline, and centrifuged at 1000g at room temperature for 10 min.

Resuspending the cells in 10mL RPMI1640 medium, counting, and taking 4X 10 ⁶ The immune cells are used for preparing dendritic cells (DC cells); and centrifuging the rest immune cell suspension, collecting immune cells, and freezing for later use.

S2 preparation of dendritic cells (i.e., DC cells)

And (3) contacting immune cells with a Tax protein of the STLV4 virus, and preparing dendritic cells after cell culture. The preparation method comprises the following steps:

s21, transfer 4X 10 ⁶ Fresh immune cells prepared in S1 were plated in one well of a 6-well plate in 10wt% FBS + RPMI1640, and cells were stimulated and cultured by adding 5. mu.g/mL PHA +100unit/mL IL 2; after 3 days of stimulation, the single cells suspended in the wells were aspirated along with the medium and discarded, leaving the clumped cells.

S22, transferring the cells into a sterile centrifuge tube, adding RPMI1640 medium to resuspend the cells, counting, and performing 2X 10 cell counting ⁶ The cells are inoculated into a new 6-well plate, and STLV4 Tax lentivirus with the MOI of 20 and glucan of 10 microgram/mL are added into the hole for co-culture for 6 hours; the cells were collected, centrifuged, and the supernatant discarded. Among them, STLV4 Tax lentivirus was prepared from lentivirus preparation example 1.

S23, resuspending the cells in RPMI1640 medium containing 10wt% serum, and adding 50unit/mL IL2 to continue culturing the cells; and the culture was continued by changing the fresh medium and 50 units/mL of IL2 every 3 days.

S24, 15 days after lentivirus transduction, cells were counted and centrifuged at 1500rpm for 5min and the supernatant was discarded. Then washing once with 5mL PBS buffer solution, centrifuging for 5min at 1500rpm, and removing the supernatant; 500. mu.L of RPMI1640 medium and a corresponding amount of anti-CD 3 magnetic beads were added and incubated for 15min at room temperature.

S25, adding 5mL of PBS buffer solution into the obtained cell culture solution in S24, reversing the solution from top to bottom, uniformly mixing the solution, placing the solution on a magnetic frame for standing for 2min, transferring cells which are not connected to magnetic beads under the action of magnetic force, placing the cells into a 6-well plate, and continuously culturing the cells in a culture medium containing 50unit/mL of IL2 and RPMI1640 until enough cell populations with adherent growth are obtained.

S26, adding pancreatin digestion cells into the cells obtained by culture, collecting the digested cells for flow cytometry analysis, detecting the expression of molecules such as CD83, CD80, CD86, CD70, CCR7, 4-1BBL and HLA-DR, and confirming the phenotype of adherent cells. The specific detection results are shown in fig. 1: of the 4 tDCs obtained, three of them grew at a relatively slow moderate growth rate in the medium, while one of them grew at a moderately fast growth rate. the typical immunophenotype of the tDC cells is shown below, indicating that these cells are derived from the myeloid lineage (CD 11c +) and exhibit their maturation (CD 83 +) and activation phenotypes (HLA-DR +/CCR7+/CD80+/CD86 +). In addition, more than 80% of DC cells show CD141 positivity, and the DC cells have strong potential of cross presenting soluble antigen and are the most important DC cells for mediating the anti-tumor and anti-virus effects of CD8+ T cells in human bodies.

S3, mixing immune cell prepared in step S1 with dendritic cell, and culturing to obtain T in cell culture _γδ A cell, the preparation comprising the steps of:

s31, continuously amplifying the dendritic cells prepared from S2, freezing and preserving seeds and amplifying T _γδ A cell;

recovering S1 frozen immune cells, counting according to 2 × 10 ⁶ The solution is inoculated into a 6-hole plate in a volume ratio of one mL to another, and 5wt% of human serum and RPMI1640 are added into the culture medium;

s32, following dendritic cell: adding dendritic cells into the immune cells according to the quantity ratio of the immune cells =1:300, uniformly mixing, and then placing in an incubator for overnight culture; the next day, adding 800unit/mL IL2 into the wells, mixing and culturing; and according to the growth rate of the cells, a culture medium containing human serum and IL2 are supplemented; the cells were harvested at day 18-21 and the cell fraction was analyzed by flow analysis.

The specific results are shown in FIG. 2: as a result, it was found that 95% or more of the cells in the prepared dendritic cells were T cells, and about 80% of them were T cells _γδ Cells (i.e., TCR γ δ +). These T _γδ The cells express markers typical of antigen presenting cells, such as co-stimulatory molecules like CD80, CD86, etc.; also expresses the NK cell activation receptor NKG 2D; and the cytotoxic T cell marker TRAIL.

Example 2

The present embodiment is different from embodiment 1 in that the operation of step S23 is: the cells were resuspended in RPMI1640 medium containing 10wt% serum and continued to be cultured by adding 200 units/mL IL2 and replaced daily for the first 2 weeks. Otherwise, the same procedure as in example 1 was repeated.

The experimental results are as follows: the culture medium turned yellow more easily than in example 1, indicating vigorous cell growth. However, after 2 weeks, the cell growth rate was significantly reduced, a large number of dead cells appeared, and the overall state of the cells was inferior to that of example 1, resulting in a lower success rate of finally preparing such DC cells than that of example 1.

Example 3

This example differs from example 1 in that in step S22, dextran was replaced with polybrene of equal weight; the method specifically comprises the following steps:

s22, transferring the cells into a sterile centrifuge tube, adding RPMI1640 medium to resuspend the cells, counting, and performing 2X 10 cell counting ⁶ Inoculating cells into a new 6-well plate, adding STLV4 Tax lentivirus with the MOI of 20 and polybrene with the MOI of 10 mug/mL into the hole, and co-culturing for 6 h; cells were collected, centrifuged, and the supernatant discarded. Among them, STLV4 Tax lentivirus was prepared from lentivirus preparation example 1.

Otherwise, the same procedure as in example 1 was repeated.

The experimental results are as follows: after lentivirus transfection, the cell status was not significantly different compared to example 1. However, the method of example 1 is about 10% higher than example 3 in terms of the overall success rate of preparing expandable dendritic cells.

Application example

Application example 1

T prepared in example 1 of the present application _γδ When the cell is used for inhibiting the cervical cancer cell HeLa, the method comprises the following steps:

(1) 1-2 × 10 parts of cervical cancer cell HeLa ⁵ Inoculating each cell/well into 6-well plate, adding RPMI1640 culture medium containing 10% FBS, and standing for 3 hr;

(2) the T prepared in example 1 _γδ Adding cells (namely effector cells) into target cell pores according to the effective target ratio of 0:1, 2.5:1, 5:1 and 10:1, and continuously culturing for 16 h;

(3) the culture medium supernatant is aspirated and discarded, and after the cells in the wells are washed once with 2mL of phosphate buffer (PBS buffer), the PBS buffer is discarded;

(4) repeating the step (3);

(5) samples were taken and the killing activity was observed by microscope and photographed, and the result of microscopic observation is shown in FIG. 3-1. The results of measuring the luciferase activity in the viable cells after cell lysis and calculating the cell viability, which are shown in FIGS. 3-2. FIG. 3 illustrates T prepared in example 1 at an effective target ratio of 10:1 _γδ The cell has excellent killing effect on the cervical cancer cell HeLa, and the cell survival rate is lower than 5%.

Application example 2

Application example 1 in contrast, T prepared by the present application _γδ The cells were used to kill melanoma cells A375, as described in example 1.

The microscopic observation result thereof is shown in FIG. 4-1. The results of measuring the luciferase activity in the viable cells after cell lysis and calculating the cell viability are shown in FIGS. 4-2. FIG. 4 illustrates T prepared in example 1 at an effective target ratio of 10:1 _γδ The cells have excellent killing effect on melanoma cells A375, and the cell survival rate of the cells is lower than 5%.

The specific embodiments are only for explaining the present application and are not limiting to the present application, and those skilled in the art can make modifications to the embodiments without inventive contribution as required after reading the present specification, but all the embodiments are protected by patent law within the scope of the claims of the present application.

Sequence listing

<110> Beijing assist Bopu Biotechnology development Limited

<120> tonsil-derived T gamma delta cell and preparation method and application thereof

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<170> SIPOSequenceListing 1.0

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atggcacatt tccccggttt cggacagagt ctcctgtacg gatatcctgt ttatgtgttc 60

ggcgactgtg tacaggccga ttggtgtcca atcagtgggg gactttgtag tcccagactg 120

caccggcatg cactcctcgc gacatgcccc gaacaccaga tcacgtggga ccctatcgac 180

ggtcgggtgg ttgggtctcc actgcagtat ttgataccca ggttgccttc atttccgaca 240

cagaggacaa gcaagaccct caaagtgctt acccctccaa ctactccagt cacgcctaag 300

gtgcctccaa gctttttcca atctgttcgc cgacatagcc cgtaccgcaa tggctgcctc 360

gagactacgt tgggggagca gctgccatct ttggccttcc cggaacccgg cctcaggccc 420

caaaatgttt acaccatttg gggtaagaca atcgtctgcc tgtacatata tcagctgagt 480

ccccctatga catggccact gatcccacac gtaatcttct gcaaccccag acaactcggg 540

gcattcctct ccaacgtgcc acctaagagg ctggaagagc tcctttataa actctacctg 600

cacacaggtg ccattattat cctccccgaa gacgcactgc cgacgacttt gttccaacct 660

gtgcgagccc catgcgtcca gaccacatgg aacacaggtc tgctccccta ccagcccaac 720

ctgacaaccc caggcctcat ttggacattt aacgatggct cccccatgat atccggtcct 780

tgtcctaagg caggtcagcc cagcctggtt gtgcagagct ccctcctgat cttcgagaga 840

ttccagacta aagcatatca tccatcatac ctgctctccc accaactcat ccagtattca 900

agtttccatc acctctatct gctctttgac gaatacacca ccattccctt tagtctcctc 960

ttcaaggaaa aagagggaga cgacagggat aatgatccac tgccaggagc aacagcgagc 1020

ccccagggcc aaaac 1035

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Met Ala His Phe Pro Gly Phe Gly Gln Ser Leu Leu Tyr Gly Tyr Pro

1 5 10 15

Val Tyr Val Phe Gly Asp Cys Val Gln Ala Asp Trp Cys Pro Ile Ser

20 25 30

Gly Gly Leu Cys Ser Pro Arg Leu His Arg His Ala Leu Leu Ala Thr

35 40 45

Cys Pro Glu His Gln Ile Thr Trp Asp Pro Ile Asp Gly Arg Val Val

50 55 60

Gly Ser Pro Leu Gln Tyr Leu Ile Pro Arg Leu Pro Ser Phe Pro Thr

65 70 75 80

Gln Arg Thr Ser Lys Thr Leu Lys Val Leu Thr Pro Pro Thr Thr Pro

85 90 95

Val Thr Pro Lys Val Pro Pro Ser Phe Phe Gln Ser Val Arg Arg His

100 105 110

Ser Pro Tyr Arg Asn Gly Cys Leu Glu Thr Thr Leu Gly Glu Gln Leu

115 120 125

Pro Ser Leu Ala Phe Pro Glu Pro Gly Leu Arg Pro Gln Asn Val Tyr

130 135 140

Thr Ile Trp Gly Lys Thr Ile Val Cys Leu Tyr Ile Tyr Gln Leu Ser

145 150 155 160

Pro Pro Met Thr Trp Pro Leu Ile Pro His Val Ile Phe Cys Asn Pro

165 170 175

Arg Gln Leu Gly Ala Phe Leu Ser Asn Val Pro Pro Lys Arg Leu Glu

180 185 190

Glu Leu Leu Tyr Lys Leu Tyr Leu His Thr Gly Ala Ile Ile Ile Leu

195 200 205

Pro Glu Asp Ala Leu Pro Thr Thr Leu Phe Gln Pro Val Arg Ala Pro

210 215 220

Cys Val Gln Thr Thr Trp Asn Thr Gly Leu Leu Pro Tyr Gln Pro Asn

225 230 235 240

Leu Thr Thr Pro Gly Leu Ile Trp Thr Phe Asn Asp Gly Ser Pro Met

245 250 255

Ile Ser Gly Pro Cys Pro Lys Ala Gly Gln Pro Ser Leu Val Val Gln

260 265 270

Ser Ser Leu Leu Ile Phe Glu Arg Phe Gln Thr Lys Ala Tyr His Pro

275 280 285

Ser Tyr Leu Leu Ser His Gln Leu Ile Gln Tyr Ser Ser Phe His His

290 295 300

Leu Tyr Leu Leu Phe Asp Glu Tyr Thr Thr Ile Pro Phe Ser Leu Leu

305 310 315 320

Phe Lys Glu Lys Glu Gly Asp Asp Arg Asp Asn Asp Pro Leu Pro Gly

325 330 335

Ala Thr Ala Ser Pro Gln Gly Gln Asn

340 345

Claims

1. Tonsil-derived T _γδ A method for producing a cell, comprising the steps of:

s1, separating immune cells from the tonsil;

s3, mixing immune cell prepared in step S1 with dendritic cell, and culturing to obtain T in cell culture _γδ A cell;

in the step S2, when the Tax protein of the STLV4 virus is contacted with immune cells, 5-15 mug/mL of glucan is added;

80% -95% of the dendritic cells prepared in the step S2 are CD141+ subtypes.

2. The preparation method according to claim 1, wherein the gene sequence encoding the Tax protein is shown as SEQ ID number 1, and the amino acid sequence of the Tax protein is shown as SEQ ID number 2.

3. The method according to claim 1, wherein in step S2, 30-80 unit/mL of IL2 is added during cell culture, and 30-80 unit/mL of IL2 is added after changing the culture medium every 2-4 days.

4. The method according to claim 1, wherein the step of separating the immune cells from the tonsil in step S1 comprises the steps of cutting the tonsil into pieces, sieving with a 175-225 mesh sieve under the action of external force to obtain dispersed cells, and centrifuging the dispersed cells with Ficoll-Hypaque solution.

5. The method according to claim 4, wherein the Ficoll-Hypaque centrifugation comprises the steps of:

6. Tonsil-derived T _γδ A cell characterized by said T _γδ The cell is prepared by the preparation method of any one of claims 1 to 5.

7. T according to claim 6 _γδ A cell characterized by said T _γδ Cell-expressed markers include markers commonly found in antigen presenting cells such as CD80 and CD86, NK cell activation receptor NKG2D, and the cytotoxic T cell marker TRAIL.

8. Tonsil-derived T _γδ Use of cells for the preparation of an antitumor or antiviral cell product, characterized in that said T is _γδ The cell is prepared by the preparation method of any one of claims 1 to 5.