CN114703134A

CN114703134A - Method for differentiating and amplifying Th17 cells in vitro and application

Info

Publication number: CN114703134A
Application number: CN202210268008.0A
Authority: CN
Inventors: 徐阳; J·Y·李
Original assignee: Laitekang Suzhou Biopharmaceutical Co ltd
Current assignee: Laitekang Suzhou Biopharmaceutical Co ltd
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2022-07-05

Abstract

The invention provides a method for differentiating and amplifying Th17 cells in vitro and application thereof. The invention also provides compositions and methods for selectively differentiating and expanding Th17 cells in vitro. The method and the composition provided by the invention not only have sufficient quantity and high homogeneity of Th17 cells, but also have the characteristics of high differentiation, high stability and strong functional activity; the method is simple, easy to operate and low in cost. The Th17 cell generated by the invention is suitable for researching the molecular mechanism of the immune regulation and the immune balance of the body and detecting the biological function of the therapeutic drug for diseases or symptoms related to the abnormal activation of the Th17 cell.

Description

Method for differentiating and amplifying Th17 cells in vitro and application

Technical Field

The invention belongs to the field of cell culture, and relates to a method for differentiating and amplifying Th17 cells in vitro and application thereof. In particular, the present invention relates to compositions and methods for selectively differentiating and expanding Th17 cells in vitro, and to Th17 cells capable of producing high homogeneity by the methods of the invention.

Background

CD4⁺Helper T lymphocytes (Th) are mediators of cellular immunity and play a key role in activating other immune cells (e.g., B cells and cytotoxic T cells) and regulating immune responses. Unoriented differentiated CD4⁺Th cells are called naive (naive) CD4⁺T cells or Th precursors (Th0), after recognition of antigen, initiate CD4⁺T cells undergo clonal expansion and differentiate into different effector cell subtypes (i.e., CD 4) under the influence of different cytokines, transcription factors, and costimulatory signals and adhesion molecules⁺Th cell subset) of CD4⁺Th cell subsets including Th1, Th2, Th17 and regulatory T cells (Tregs),each subgroup is activated by a group of specific cytokines and transcription factors, and plays different effect functions through secreting different cytokines, and the cytokines are mutually regulated and controlled, so that the organism is in a fine and complex immune balance state and plays an important role in cellular immunity.

Th17 cells were the third category of effector CD4 following the discovery of Th1 and Th2 cells⁺Th cell subsets, named for their high level of IL-17 secretion. Th17 mainly secretes cytokines such as IL-17A, IL-17F, IL-21 and IL-22, expresses transcription factor retinoic acid-Related Orphan Receptor (ROR) gt and RORa, and plays a role in host defense against extracellular pathogens, particularly mucosa and epithelial barriers, but Th17 cell dysregulation is related to the occurrence of various autoimmune diseases (including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, psoriasis and the like).

Currently, Th17 cells have become a hot research spot in the field of immunology, and more researches have been conducted to explore Th17 cells and cytokine products thereof as therapeutic targets of various autoimmune diseases, so that obtaining a large number of Th17 cells with high homogeneity and stability in vitro becomes a first condition for researching Th17 cells. However, the difference in the in vitro differentiation and expansion method often results in great difference in the obtained Th17 cells, which are shown by insufficient number, poor homogeneity and/or instability, and are easy to transdifferentiate or dedifferentiate.

Therefore, it is highly desirable to develop a method for differentiating and expanding in vitro to obtain a sufficient number of Th17 cells with high homogeneity and stability, which is suitable for the research of the molecular mechanisms of immune regulation and balance and the detection of the biological functions of the therapeutic drugs for diseases or disorders associated with abnormal activation of Th17 cells.

Disclosure of Invention

In view of the technical defects of the prior art, the invention provides a method for differentiating and expanding Th17 cells in vitro, which solves the problem of primary CD4⁺The urgent need for differentiation and expansion of T cells into Th7 cells of sufficient number, high homogeneity and stability. The invention also relates toCompositions for selectively differentiating and expanding Th17 cells in vitro and methods for producing Th17 cells are provided. The method and the composition provided by the invention can generate sufficient Th17, and the Th17 has the characteristics of good cell activity, high homogeneity, high differentiation, high stability and strong functional activity; the method is simple, easy to operate and low in cost; the Th17 cell generated by the invention is suitable for detecting the biological activity/function of the therapeutic drug for diseases or symptoms related to Th17 cell disorder and/or for researching immunological mechanisms (such as molecular mechanisms of body immune regulation and immune balance).

In one aspect, the present invention provides a method for differentiating and expanding Th17 cells in vitro, comprising the steps of:

(1) obtaining an initial CD4⁺T cell: the initial CD4⁺T cells are selected from CD4⁺CD45RA⁺A T cell;

(2) activating, differentiating and expanding the cells in the step (1) into Th17 cells in a cell culture medium;

(3) harvesting the Th17 cells obtained in step (2) and detecting.

The initial CD4 in the step (1)⁺Sources of T cells include, but are not limited to, human peripheral blood, cord blood, blood cells within immune organs (e.g., spleen or lymph nodes), and blood precursor cells. Initial CD4⁺T cells include undifferentiated T cells expressing the surface antigen of CD4, including but not limited to human peripheral blood, cord blood, and other previously manipulated CD4⁺T cells. T cell precursors such as hematopoietic stem cells and lymphoblast cells obtained from bone marrow, umbilical cord blood, etc. can be induced to differentiate into CD4⁺T cells.

The initial CD4⁺The T cells can be separated and purified from the monocyte group in the source by using a magnetic bead separation technology, and can also be commercialized separated and purified primary human CD4⁺CD45RA⁺T cells.

The cell culture medium in step (2) comprises a basal cell culture medium and additives comprising a polarizing cytokine (polarizing cytokine) and IL-2, wherein the polarizing cytokine comprises one or more of TGF-b, IL-1b, IL-6, IL-21 and IL-23, preferably the polarizing cytokine is TGF-b, IL-1b, IL-6 and IL-23.

Further, the concentration of TGF-b in the cell culture medium is 1-10ng/ml, preferably 2.5-10ng/ml, more preferably 2.5 ng/ml; the concentration of IL-6 in the cell culture medium is 10-50ng/ml, preferably 30 ng/ml.

The additive TGF-b can directly induce and cooperate with IL-6 to promote the expression of RORgt and RORa through STAT3 signals, so that the CD4 is initially⁺The directional differentiation of T cells into Th17 cells; at the same time, the presence of IL-6 and its activated STAT3 signal favoured the downregulation of the initial CD4⁺T cells express Foxp3, inhibiting TGF-b induction of CD4⁺T cells differentiate into Treg cells. The inventors have unexpectedly found that when the concentration ratio of IL-6 to TGF-b is maintained at 5-12, the initial CD4⁺The T cells can remarkably up-regulate the expression quantity of RORgt and maintain the high level, thereby ensuring the initial CD4⁺T cells can maximally differentiate to Th17 cells, and the differentiation degree is high. Further, IL-1b in the supplement is not only capable of stimulating primary CD4⁺The T cells can differentiate to Th17 cells, and can also enhance the inhibition effect of IL-6 on TGF-b induced Treg cell differentiation, thereby being beneficial to the initial CD4⁺T cells differentiated into Th17 cells. IL-23 can promote maturation, survival and proliferation of Th17, is beneficial to high expression and IL-17 secretion of Th17, and also has the potential of reducing dedifferentiation of Th 17.

According to the invention, a large number of experiments show that when TGF-b, IL-1b, IL-6, IL-23 and IL-2 are simultaneously added into a basic cell culture medium and are controlled within the concentration range, the TGF-b, the IL-1b, the IL-6, the IL-23 and the IL-2 have a synergistic effect on maximally promoting the differentiation of Th17 cells, and the survival, the amplification, the effector function and the stability of Th17 cells can be remarkably maintained. Wherein, preferably, the concentration of IL-1 beta in the cell culture medium is 20ng/mL, the concentration of IL-23 in the cell culture medium is 30ng/mL, and the concentration of IL-2 in the cell culture medium is 10-30U/mL.

The supplement of step (2) may further comprise anti-CD 3 antibody and anti-CD 28 antibody, and the anti-CD 3 antibody and anti-CDThe 28 antibody may be soluble or in the form of immunomagnetic beads. The anti-CD 3 and anti-CD 28 antibodies and the IL-2 are effective in activating and/or amplifying primary CD4⁺T cells, favouring the priming of CD4⁺T cells differentiated into Th17 cells.

The basic cell culture medium in step (2) may be serum-free cell culture medium, or may be cell culture medium containing serum, wherein the serum is 10-20% Fetal Bovine Serum (FBS).

The serum-free cell culture medium is selected from one or more of the following commercial culture media: yssel's T Cell Medium, Immunocult T Cell Medium, CTS OpTsizer T Cell expansion SFM and AIM V Medium, preferably Yssel's T Cell Medium, Immunocult T Cell Medium, more preferably Immunocult T Cell Medium.

The inventors of the present invention unexpectedly found that Th17 cells cultured in a serum-containing cell culture medium exhibited better cell numbers, cell survival rates, RORgt expression levels, and IL-17 secretion levels than serum-free cell culture medium.

Culturing the cells in the step (2) in the cell culture medium for at least 3 days, preferably 6 days, and more preferably, separating the cells after culturing the cells in the cell culture medium for 3 days and adding fresh cell culture medium again to continue culturing to the 6 th day.

The inventors surprisingly found that the initial CD4 is not adversely affected⁺T cells were cultured for 6 days with continued activation, differentiation and expansion, with naive CD4 cultured in cell culture medium comprising the polarized cytokine, IL-2 and anti-CD 3 and anti-CD 28 antibodies as compared to day 0⁺T cells, and cells obtained by culturing the cells without the anti-CD 3 antibody and anti-CD 28 antibody at day 3 to day 6, were replaced not only so that the original CD4 was obtained⁺Differentiation of T cells into CCR6 with terminal differentiation effector phenotype⁺Th17 cells, and sufficient Th17 cells with high homogeneity and strong cell function activity are obtained.

The additive in step (2) may further comprise anti-IFN-g antibody and anti-IL-4 antibody, which are effective in preventingTh17 differentiation was inhibited, and primary CD4 was prevented⁺T cells to other CD4⁺Th cells (e.g., Th1 and Th2) differentiate.

The detection in the step (3) comprises a surface marker or cell surface antigen of the Th17 cell, a transcription cytokine and a secreted cytokine. The surface markers or cell surface antigens include CD39, CD121a, CD161, CCR4, CCR6, CXCR3, IL-21R, and IL-23R, preferably CCR6 and CXCR 3; the transcription factor comprises RORgt or RORa; the secreted cytokines include IL-17A, IL-17AF, IL-17F, IL-21, and IL-22, preferably IL-17A.

In another aspect, the present invention provides a composition for selectively differentiating and expanding Th17 cells in vitro and a method for producing Th17 cells using the same. The compositions comprise polarized cytokines and IL-2, wherein the polarized cytokines comprise one or more of TGF-b, IL-1b, IL-6, IL-21, and IL-23, preferably the polarized cytokines are TGF-b, IL-1b, IL-6, and IL-23. The composition may further comprise anti-CD 3 and anti-CD 28 antibodies and/or anti-IFN-g, anti-IL-4 antibodies. The method comprises the following steps: (1) acquisition of primary CD4+ T cells: the initial CD4⁺The T cell is CD4⁺CD45RA⁺A T cell; (2) adding the composition into a basic cell culture medium to prepare the cell culture medium of the invention, and activating, differentiating and expanding the cells in the step (1) into Th17 cells in the cell culture medium, wherein the concentration of TGF-b in the composition in the cell culture medium is 1-10ng/ml, preferably 2.5-10ng/ml, and more preferably 2.5 ng/ml; the concentration of IL-6 in the cell culture medium is 10-50ng/ml, preferably 30 ng/ml; the concentration ratio of the IL-6 to the TGF-beta is 5-12. Further, the composition further comprises IL-1 beta, IL-23 and IL-2, wherein the concentration of IL-1b in the cell culture medium is 20 ng/ml; the concentration of IL-23 in the cell culture medium was 30 ng/ml; the concentration of IL-2 in the cell culture medium is 10-30U/ml. The basic cell culture medium can be a serum-free cell culture medium, and can also be a cell culture medium containing 10-20% FBS; (3) harvesting the Th17 cells obtained in step (2) and detecting surface markers or cell surface antigens, transcription factors and secreted cytokines of the Th17 cellsAnd (4) adding the active ingredients.

In a third aspect, the invention provides a use of the Th17 cell obtained by the method and the composition in researching immunological mechanism and/or detecting biological function of a therapeutic drug for diseases or symptoms related to Th17 cell dysregulation. Such diseases or conditions associated with Th17 cell dysregulation include, but are not limited to, autoimmune diseases (including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, psoriasis, and the like) and other atopic and chronic inflammatory diseases.

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

1. by screening additives (comprising cytokines, FBS, anti-CD 3 antibody and anti-CD 28 antibody) added into a basic cell culture medium and performing a series of optimization on the concentration or proportion of the cytokines, the concentration of the FBS and a culture mode, not only can enough number of Th17 cells with good vitality and high homogeneity be generated, but also the obtained Th17 cells have a terminal differentiation effect phenotype and high stability and are not easy to transdifferentiate into other CD4 cells⁺Helper T subtype and dedifferentiation to primary CD4⁺A T cell;

2. the composition has simple components and reasonable proportion among the components, and the Th17 cells generated by adding the composition into a basic cell culture medium are not only sufficient in quantity, good in activity and high in homogeneity, but also terminally differentiated, high in stability and not easy to transdifferentiate or dedifferentiate;

3. the method and the composition for differentiating and amplifying the Th17 cells in vitro and the method for generating the Th17 cells are provided, the method has strong applicability, good repeatability and stability, low cost, simple method and easy operation;

4. through a large number of research experiments, it is found that if the cells are cultured for 6 days without changing the culture medium or are changed to a fresh cell culture medium without anti-CD 3 and anti-CD 28 on day 3, the T cells become depleted because of not being continuously activated and differentiating-promoted so as not to secrete cytokines to enhance cell differentiation and are not easily reactivated, and thus the effect of the method of the present invention is difficult to achieve;

5. provided are a method and a composition for differentiating and expanding Th17 cells in vitro and a method for generating Th17 cells, wherein the Th17 cells obtained by the method are suitable for researching immunological mechanism and/or detecting biological functions of a medicine for treating diseases or symptoms related to Th17 cell disorder.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments.

Term interpretation (Chinese name corresponding to English code of the invention)

Serial number	English	Chinese character
			1	helper T cell,Th	Helper T lymphocytes
2	transforming growth factor-β,TGF-b	Transforming growth factor-b
			3	interleukin,IL	Interleukins
4	regulatory T cells,Treg	Regulatory T cells
			5	retinoid-related orphan receptor,ROR	Retinoic acid related orphan nuclear receptors
6	Forkhead box protein P3,FOXP3	Bifurcate box protein P3
			7	fetal bovine serum,FBS	Fetal bovine serum
8	peripheral blood mononuclear cells,PBMC	Peripheral blood mononuclear cells
			9	Median Fluorescence Intensity,MFI	Mean fluorescence intensity

Drawings

FIG. 1 human Primary CD4 under different conditions⁺T cell differentiation and expansion into cell viability (a) and viable cell density (b) of Th17 cells on day 6, wherein Stemcell IC 6 indicates that anti-CD 3 and anti-CD 28 antibodies contained in the cell culture medium were ImmunoCult from STEMCELL^TMHuman CD3/CD 28T Cell Activator, and the respective Cell media shown in Table 1 were added on day 0 and day 3 in the culture manner of example 1, and were co-cultured6 days; stemcell IC 3/3 shows that the anti-CD 3 and anti-CD 28 antibodies contained in the cell culture medium were ImmunoCult from STEMCELL^TMHuman CD3/CD 28T Cell Activator, and each Cell culture medium shown in Table 1 was added on day 0 using the culture method of the comparative example, the cells were separated on day 3, and fresh medium without anti-CD 3 and anti-CD 28 was added to continue the culture until day 6; dynabeads 6 indicates that the anti-CD 3 and anti-CD 28 antibodies contained in the cell culture medium are

Human T-Activator CD3/CD28, and each cell culture medium shown in Table 2 was added on day 0 and day 3 in the culture manner of example 1 for a total of 6 days; dynabeads 3/3 indicates that the anti-CD 3 and anti-CD 28 antibodies contained in the cell culture medium are

Human T-Activator CD3/CD28, and each cell culture medium shown in Table 2 was added on day 0 using the culture method of the comparative example, the cells were separated on day 3, and fresh medium without anti-CD 3 and anti-CD 28 was added to continue the culture until day 6.

FIG. 2 flow cytometry detection of human Primary CD4⁺RORgt obtained at day 6 after T-cell differentiation and expansion under different conditions⁺CD45RO⁺Wherein Stemcell IC 6 represents that the anti-CD 3 and anti-CD 28 antibodies contained in the cell culture medium are ImmunoCult from STEMCELL^TMHuman CD3/CD 28T Cell Activator, and each Cell culture medium shown in Table 1 was added on day 0 and day 3 in the culture manner of example 1 for a total of 6 days; stemcell IC 3/3 shows that the anti-CD 3 and anti-CD 28 antibodies contained in the cell culture medium were ImmunoCult from STEMCELL^TMHuman CD3/CD 28T Cell Activator, and each Cell culture medium shown in Table 1 was added on day 0 using the culture method of the comparative example, cells were separated on day 3, and fresh medium without anti-CD 3 and anti-CD 28 was added to continue culturing until day 6; dynabeads 6 indicates that the anti-CD 3 and anti-CD 28 antibodies contained in the cell culture medium are

Human T-Activator CD3/CD28, and each cell culture medium shown in Table 2 was added on day 0 and day 3 in the culture manner of example 1 for 6 days in total; dynabeads 3/3 indicates that the anti-CD 3 and anti-CD 28 antibodies contained in the cell culture medium are

FIG. 3 human Primary CD4 under different conditions⁺T cells were differentiated and expanded into RORgt expression levels of Th17 cells on day 6, wherein Stemcell IC 6 indicates that anti-CD 3 and anti-CD 28 antibodies contained in the cell culture medium were ImmunoCult from STEMCELL^TMHuman CD3/CD 28T Cell Activator, and each Cell culture medium shown in Table 1 was added on day 0 and day 3 in the culture manner of example 1 for a total of 6 days; stemcell IC 3/3 shows that the anti-CD 3 and anti-CD 28 antibodies contained in the cell culture medium were ImmunoCult from STEMCELL^TMHuman CD3/CD 28T Cell Activator, and each Cell culture medium shown in Table 1 was added on day 0 using the culture method of the comparative example, the cells were separated on day 3, and fresh medium without anti-CD 3 and anti-CD 28 was added to continue the culture until day 6; dynabeads 6 indicates that the anti-CD 3 and anti-CD 28 antibodies contained in the cell culture medium are

Human T-Activator CD3/CD28, and each cell culture medium shown in Table 2 was added on day 0 in the manner of culture of comparative example,cells were isolated on day 3 and culture continued to day 6 by adding fresh medium without anti-CD 3 and anti-CD 28.

FIG. 4 shows that the differentiation and expansion of human primary CD4+ T cells into Th17 cells at day 6 IL-17A secretion by AlphaLISA method, wherein Stemcell IC 6 indicates that the anti-CD 3 and anti-CD 28 antibodies contained in the cell culture medium are ImmunoCult from STEMCELL^TMHuman CD3/CD 28T Cell Activator, and each Cell culture medium shown in Table 1 was added on day 0 and day 3 in the culture manner of example 1 for a total of 6 days; stemcell IC 3/3 shows that the anti-CD 3 and anti-CD 28 antibodies contained in the cell culture medium were ImmunoCult from STEMCELL^TMHuman CD3/CD 28T Cell Activator, and each Cell culture medium shown in Table 1 was added on day 0 using the culture method of the comparative example, the cells were separated on day 3, and fresh medium without anti-CD 3 and anti-CD 28 was added to continue the culture until day 6; dynabeads 6 indicates that the anti-CD 3 and anti-CD 28 antibodies contained in the cell culture medium are

FIG. 5 flow cytometry detection of G1 group containing anti-CD 3 and anti-CD 28 soluble antibodies in cell culture media ImmunoCult containing anti-CD 3 and anti-CD 28 soluble antibodies was added on

days

0 and 3^TMHuman CD3/CD 28T Cell Activator (denoted G1 Stemcell IC 6) and Cell culture medium G5 group (denoted G5 Dynabeads 6) differentiation and differentiation of Cell culture medium comprising anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads, IL-2 and polarized cytokinesExpanded to the degree of differentiation of Th17 cells at day 6, cell surface antigen CXCR3⁺CCR6⁺Th17 cells.

Detailed Description

The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, patents and patent applications cited throughout this application are expressly incorporated herein by reference.

EXAMPLE 1A method for differentiating and expanding Th17 cells

1. Isolation of purified human Primary CD4 from human peripheral blood⁺T cells

Collecting peripheral blood of healthy people, performing Ficoll density gradient treatment to obtain Peripheral Blood Mononuclear Cells (PBMC), and separating from PBMC with magnetic bead separation technology to obtain purified human primary CD4⁺T cells, i.e. primary human CD4⁺CD45RA⁺T cells.

2. Differentiation and expansion of Th17 cells

(1) Preparation of cell culture Medium

Different cell culture media for differentiation and expansion into Th17 cells were prepared by adding different concentrations of cytokines, antibodies and/or FBS to the basal medium according to the formulations of tables 1 and 2. Wherein the basal Medium comprises RMPI (containing 2mM glutamine, 55. mu.M BME, 25mM HEPES, 1mM sodium pyruvate and 0.1mM non-essential amino acids), Yssel's T Cell Medium (Cat. 900-103, Gemini Bioproducts Co., Ltd.) and ImmunoCult^TMT cell extension medium (cat # 10981, STEMCELL Co.). Table 1 shows that anti-CD 3 antibody and anti-CD 28 antibody were used in each group of cell culture media, and ImmunoCult manufactured by STEMCELL corporation was used^TMHuman CD3/CD 28T Cell Activator (cat # 10971) for activating Human primary CD4 prepared in step 1⁺A T cell; table 2 shows that the anti-CD 3 antibody and the anti-CD 28 antibody used in each cell culture medium were those produced by Gibco "

Human T-Activator CD3/CD 28' to activate the Human original CD prepared in step 14⁺T cells.

TABLE 1

TABLE 2

(2) Differentiation and expansion of Th17 cells

Grouped according to tables 1 and 2, the human primary CD4 obtained in step 1 above was suspended in cell culture media prepared in tables 1 and 2, respectively⁺Mixing T cells, adjusting cell density to 1 × 10⁶Perml, and added to a 96-well plate at a volume of 250mL per well, and incubated in a carbon dioxide incubator saturated with 5% CO2 humidity at 37 ℃. On day 3, after counting the cells, the cells were centrifuged, the supernatant was removed, the cell culture media prepared in tables 1 and 2 were added, respectively, and the mixture was placed in a carbon dioxide incubator saturated with 5% CO2 humidity at 37 ℃ for further culture up to day 6 (this culture mode is referred to as continuous activation, differentiation and amplification culture for 6 days). On day 6, the cells were counted, the cell viability was measured, and the results are shown in FIG. 1, in which the cell viability and the expansion ability (i.e., the number of viable cells) of the T cells of group G4 were poor in the serum cell culture medium containing the anti-CD 3 and anti-CD 28 soluble antibodies (each group shown in Stemcell 6); similarly, in the serum-free Cell culture Medium, the G6 group, whether supplemented with anti-CD 3 and anti-CD 28 soluble antibodies (each group shown in Stemcell 6) or with anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads (each group shown in Dynabeads 6), had cells with lower expansion capacity (i.e., viable Cell number) than the other groups, indicating that the basal Cell culture Medium Yssel's T Cell Medium is not suitable for Th17 Cell proliferation culture; while the concentration ratio of IL-6 to TGF-b in the other groups of cell culture media (including serum and serum-free media) was maintained at 5-12 to maintain the initial CD4⁺After the T cells are continuously activated and induced to differentiate, the T cells are clustered and rapidly proliferated, and the proliferation peak of the T cells is reached at the 6 th day (the T cells are shown to have better amplification capacity and/or be/Or cell viability rate).

In addition, a negative control group is also provided, and the method comprises the following steps: ImmunoCult in basal medium^TMIL-2 was added to the T cell expansion medium to a final concentration of 30U/mL, and the human-origin CD4 obtained in step 1 above was suspended⁺Mixing T cells, adjusting cell density to 1 × 10⁶To each well was added a volume of 250mL to a 96-well plate previously coated with an anti-CD 3 antibody (OKT3 antibody, 5. mu.g/mL) and an anti-CD 28 antibody (28.2 antibody, 2.5. mu.g/mL), and the mixture was incubated at 37 ℃ in a carbon dioxide incubator saturated with 5% CO2 humidity for 6 days.

(3) Detection assay

On day 7, supernatants were centrifuged for detection of IL-17A secretion levels and harvested cells were flow analyzed.

1) Intranuclear staining for detection of RORgt by flow cytometry

The specific method comprises the following steps:

resuspending the harvested cells in 50 mL/well of Trustain Fc block reagent-containing PBS in a 96-well format and incubating for 15 min at room temperature; then, 100 mL/well of PBS containing zymobie NIR fluorescent dye was added and incubated at room temperature for 15 minutes; washed twice with PBS; adding 100 mL/hole PBS containing surface antigen flow antibody to resuspend the cells, and incubating for 15 minutes at room temperature; washed twice with PBS; adding a fixing buffer solution, and incubating for 25 minutes at 4 ℃; resuspending the cells with a membrane breaking buffer, and incubating for 20 minutes at room temperature; centrifuging to remove supernatant, adding 50 mL/hole of membrane breaking buffer solution containing RORgt antibody for resuspending cells, and incubating for 1 hour at room temperature; washing the cells twice with a membrane breaking buffer; the proportion of CD45RO and RORgt double positive viable cells, as well as the mean Fluorescence Intensity of RORgt (Median Fluorescence Intensity, MFI) were finally examined by flow cytometry after cell resuspension with PBS.

Wherein, the detection results of the CD45RO and RORgt double-positive living cells are shown in FIG. 2 and Table 3, the proportion of the double-positive living cells obtained by differentiation and amplification in the cell culture medium group containing anti-CD 3 and anti-CD 28 soluble antibodies (each group shown in Stemcell IC 6) can reach more than 60 percent, even more than 80 percent, and is obviously higher than that of the cell culture medium containing anti-CD 3 and anti-CD 28 antibodies immunomagnetic beads Dynabeads (each group shown in Dynabeads 6)Group (d); since the concentration ratio of IL-6 to TGF- β in the cell culture medium containing anti-CD 3 and anti-CD 28 soluble antibodies (each group shown in Stemcell 6) was 2, the proportion of the number of live cells expressing ROR γ t in the G4 group was lower than that in the other groups under the same conditions, indicating that the IL-6/TGF- β concentration ratio was too low to be favorable for the initial CD4⁺T cells differentiated into Th17 cells. Although group G6 showed a low number of viable cells in Cell culture media containing anti-CD 3 and anti-CD 28 soluble antibodies (each group shown by Stemcell IC 6) and anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads (each group shown by Dynabeads 6), flow cytometry detected comparable or higher proportions of cells expressing ROR γ T relative to other groups containing the same anti-CD 3 and CD28, suggesting that although the basal Cell culture Medium Yssel's T Medium of group G6 was not an optimal choice for Th17 Cell proliferation culture, it did not affect the initial CD4 Cell proliferation culture⁺T cells differentiated into Th17 cells.

TABLE 3 detection of cell surface antigen CD45RO and proportion of cells whose nuclei express ROR γ t-positive viable cells on day 6 by flow cytometry after continuous activation, differentiation and expansion culture for 6 days

The results of flow cytometry for the expression level of RORgt are shown in fig. 3, and although each group of cell culture media containing anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads (each group shown by Dynabeads 6) had better cell expansion ability than each group of cell culture media containing anti-CD 3 and anti-CD 28 soluble antibodies (each group shown by Stemcell IC 6), the former was significantly lower than the latter in the expression level of RORgt. Since the concentration ratio of IL-6 to TGF-. beta.in the cell culture medium containing anti-CD 3 and anti-CD 28 soluble antibody (each group shown in Stemcell 6) was 2, the ROR. gamma.t expression level was lower in the G4 group than in the other groups under the same conditions, indicating that the IL-6/TGF-. beta.concentration ratio was too low to be favorable for the initial CD4⁺T cells differentiated into Th17 cells. Although group G6 contained anti-CD 3 and anti-CD 28 soluble antibodies (each group shown in Stemcell IC 6) and anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads (Dynabeads 6)Groups) but flow cytometry detected comparable or higher ROR γ T expression relative to the G7 group containing the same anti-CD 3 and CD28, further verifies that although the basal Cell culture Medium Yssel's T Cell Medium of the G6 group is not the optimal choice for proliferation culture of Th17 cells, it does not affect the initial CD4⁺T cells differentiated into Th17 cells.

2) AlphaLISA method for detecting IL-17A secretion level of Th17 cells

By using

The human IL-17(IL-17, IL17A) assay kit quantitatively detects the secretion level of IL-17A in the supernatant. The specific method comprises the following steps:

a. taking 5mL of supernatant liquid to a 96-well plate;

b. adding 20 mL/well detection mixed solution into the 96-well plate, wherein the mixed solution consists of AlphaLISA anti-IL-17A receptor magnetic beads (25mg/mL) and biotin-labeled anti-IL-17A antibodies (2.5nM), the final concentration of the AlphaLISA anti-IL-17A receptor magnetic beads in the mixed solution is 10mg/mL, and the final concentration of the biotin-labeled anti-IL-17A antibodies is 1 nM; incubating the 96-well plate for 60 minutes at room temperature;

c. 25mL of 2 XStavidin (Streptavidin, SA) -donor magnetic beads (80mg/mL) were added, with a final concentration of the SA-donor magnetic beads of 40 mg/mL; incubating for 30 minutes at room temperature in the dark;

d. and (5) placing the sample in a microplate reader to read the value of the luminescence signal.

As shown in fig. 4, the secretion amount of IL-17A of each group of cell culture media containing anti-CD 3 and anti-CD 28 soluble antibodies (each group shown in Stemcell IC 6) was significantly higher than that of each group of cell culture media containing anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads (each group shown in Dynabeads 6), and G1 group had the highest secretion amount of IL-17A of both anti-CD 3 and anti-CD 28 under the same conditions; initial human CD4 was affected when the IL-6/TGF-beta concentration ratio in the cell culture medium was < 5⁺T cells differentiate into Th17 cells, which in turn influence the secretion of IL-17A, such as group G4; furthermore, the AlphaLISA assay was based on the total fineness of each well in a 96-well plateThe amount of IL-17A secreted by the cells, and the number of viable cells in the G6 group were small in serum-free cell culture media containing anti-CD 3 and anti-CD 28 soluble antibodies (each group shown by Stemcell IC 6) and anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads (each group shown by Dynabeads 6), respectively, so that the G6 group had a significantly reduced amount of IL-17A secreted in anti-CD 3 and anti-CD 28 under the same conditions.

In summary, although each group of cell culture media comprising anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads (each group shown by Dynabeads 6) had better cell expansion ability and/or cell viability rate than each group of cell culture media comprising anti-CD 3 and anti-CD 28 soluble antibodies (each group shown by Stemcell IC 6), the former was significantly lower than the latter in terms of the RORgt expression amount and the IL-17A secretion amount; and under the same conditions, the cell culture medium group containing 10-20% FBS has higher RORgt expression amount and/or IL-17A secretion amount than the serum-free cell culture medium group (containing each group shown by Stemcell IC 6 and each group shown by Dynabeads 6); further, in the cell culture medium group (regardless of anti-CD 3 and anti-CD 28 forms) containing FBS at IL-6 to TGF-b concentration ratios maintained at 5-12 and 10-20% FBS, cell culture media containing anti-CD 3 and anti-CD 28 and each cytokine (containing IL-2 and polarized cytokines) were added on days 0 and 3, respectively, so that initial CD4⁺The Th17 cells differentiated and expanded by the T cells have good proliferation capacity, survival rate, RORgt expression activity and IL-17A secretion capacity; in addition, the serum-free cell culture medium added with IL-6 and TGF-b with the concentration ratio of 5-12 and soluble antibodies against CD3 and CD28 in the basal cell culture medium ImmunoCult can well promote the differentiation and expansion of the initial CD4+ T cells into enough number of Th17 cells with high homogeneity, ROR gamma T expression and IL-17A secretion.

Example 2 study of degree of differentiation of Th17 cells

To further investigate the degree of differentiation of the Th17 cells obtained in example 1, a G1 group (with a higher proportion of ROR γ t) containing anti-CD 3 and anti-CD 28 soluble antibodies in the cell culture medium was selected⁺Cell number and high expression of ROR γ t) and group G5 (with low proportion of ROR γ t) comprising anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads⁺Cell number and Low expressionAmount of ROR γ t), using flow cytometry as described in example 1 to detect CXCR3⁺CCR6⁺Th17 cell proportion.

As shown in FIG. 5, the results of the measurement of RORgt were obtained when the cell culture medium containing anti-CD 3 and anti-CD 28 soluble antibodies in the G1 group was cultured on

days

0 and 3 with addition of the cell culture medium containing anti-CD 3 and anti-CD 28 soluble antibodies, IL-2 and polarized cytokines to day 6⁺CXCR3 in cells⁺CCR6⁺The proportion of Th17 cells was about 58.9% (fig. 5); group G5 containing anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads in cell culture medium RORgt was cultured by adding cell culture medium containing anti-CD 3 and anti-CD 28 soluble antibodies, IL-2 and polarized cytokines to day 6 on

days

0 and 3⁺CXCR3 in the cell of (1)⁺CCR6⁺The proportion of Th17 cells was about 17.4% (FIG. 5), indicating that the RORgt obtained by differentiation and expansion using the method described in example 1⁺Th17 cell has CCR6⁺CXCR3⁺And CCR6⁺CXCR3⁺The cell ratio is positively correlated with the RORgt expression level. It was further shown that the Th17 cells obtained in example 1 were highly differentiated and stable.

In summary, human original CD4⁺The Th17 cells obtained by differentiating and amplifying the T cells in a cell culture medium containing 10-20% FBS, anti-CD 3 and anti-CD 28 soluble antibodies, IL-2 and various polarized cytokines in a reasonable concentration range, and IL-6 and TGF-beta concentrations are higher than those of the Th17 cells maintained at 5-12, and have the characteristics of good proliferation activity, high homogeneity, high degree of differentiation and high stability.

Comparative example Effect of different culture methods on differentiation and expansion of human naive CD4+ T cells into Th17 cells

1. Purified human primary CD4 was isolated from human peripheral blood using the method described in example 1⁺T cells

2. In vitro differentiation and expansion of Th17 cells

(1) Preparation of cell culture Medium

The same as in example 1.

(2) Differentiation and expansion of Th17 cells

Grouped according to tables 1 and 2, the human primary CD4 obtained in step 1 above was suspended in cell culture media formulated in tables 1 and 2, respectively⁺Mixing T cells, adjusting cell density to 1 × 10⁶and/mL, and was added to a 96-well plate at a volume of 250mL per well, placed in a carbon dioxide incubator saturated with 5% CO2 at 37 ℃ and humidity, and according to the cell differentiation and expansion, after counting the cells on day 3, the cells were centrifuged, supernatant was removed, cell culture medium without anti-CD 3 and anti-CD 28 was added, and the cells were further cultured in a carbon dioxide incubator saturated with 5% CO2 at 37 ℃ and humidity (i.e., 3 days for activation and differentiation, 3 days for non-activation for maintenance, as shown in 3/3). On day 6, the cells were counted and the cell viability was measured, and the results are shown in FIG. 1, and most of the cells in each group shown by Stemcell 3/3 were less dense than those in each group shown by Dynabeads 3/3, but in terms of cell viability, each group shown by Stemcell 3/3 was equivalent to that shown by Dynabeads 3/3; however, the cell density and viability of the groups shown in Stemcell 3/3 were mostly comparable to those of Stemcell 6, and those of Dynabeads 3/3 were also mostly comparable to those of Dynabeads 6.

(3) Detection and analysis:

the number of CD45 RO-expressing and RORgt-positive living cells, the RORgt expression level and the IL-17A secretion level obtained by the above steps were determined by the same method as described in example 1.

Expression of CD45RO⁺RORgt⁺As shown in FIG. 2 and Table 4, the results of viable cell count measurements were obtained by differentiating and amplifying CD45RO obtained by differentiation and amplification in a cell culture medium fraction containing anti-CD 3 and anti-CD 28 soluble antibodies (each fraction shown in Stemcell IC 3/3) and anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads (each fraction shown in Dynabeads 3/3)⁺RORgt⁺The proportion of viable cells was significantly lower than that shown for Stemcell IC 6 and Dynabeads 6.

TABLE 4 detection of cell surface antigen CD45RO and proportion of cells expressing ROR γ t positive viable cells by flow cytometry at day 6 using 3/3 culture

The results of flow cytometry for detecting the RORgt expression level are shown in fig. 3, and in each group of cell culture media containing anti-CD 3 and anti-CD 28 soluble antibodies, the RORgt expression level of the cells obtained by differentiation and amplification in a 3/3 culture mode is significantly lower than that of the cells obtained by co-culture for 6 days by adding the cell culture media shown in table 1 on the 0 th day and the 3 rd day respectively; in each group of cell culture media containing anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads, the cells obtained by differentiation and amplification in the 3/3 culture mode have the RORgt expression quantity equivalent to that of the cells obtained by co-culture for 6 days by respectively adding the cell culture media shown in the table 2 on the 0 th day and the 3 rd day. Furthermore, although each group of cell culture media comprising anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads (each group shown by Dynabeads 3/3) had better cell expansion ability than each group of cell culture media comprising anti-CD 3 and anti-CD 28 soluble antibodies (each group shown by Stemcell IC 3/3), the former was also found to be significantly lower than the latter in terms of the amount of RORgt expression.

The results of detecting the level of IL-17A secreted by Th17 cells by the AlphaLISA method are shown in fig. 4, and in each group containing anti-CD 3 and anti-CD 28 soluble antibody cell culture media, the IL-17A secretion of the cells obtained by differentiation and amplification in the 3/3 culture manner is significantly lower than that of the cells obtained by co-culture for 6 days by adding the cell culture media shown in table 1 on

days

0 and 3, respectively; however, in each group of the cell culture medium containing anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads, the IL-17A secretion amount of the cells obtained by differentiation and amplification in the 3/3 culture mode is equivalent to that of the cells obtained by co-culture for 6 days by adding the cell culture medium respectively shown in Table 2 on the 0 th day and the 3 rd day; furthermore, it was also found that the amount of IL-17A secretion was significantly higher in each group of cell culture media containing anti-CD 3 and anti-CD 28 soluble antibodies (each group shown by Stemcell IC 3/3) than in each group of cell culture media containing anti-CD 3 and anti-CD 28 immunomagnetic beads Dynabeads (each group shown by Dynabeads 3/3).

In summary, different culture methods were used to activate, differentiate and expand human naive CD4+ T cells into Th17 cells in RORgt under the same composition of culture medium⁺Number of viable cells, RORThe difference between the gt expression level and the IL-17A secretion level is large. It can be seen that the culture method of the invention, which adds cell culture medium containing activated proliferation-promoting anti-CD 3 and anti-CD 28 and IL-2 and differentiation-promoting polarized cytokines (including TGF-b, IL-6, IL-1b and IL-23) on day 0 and day 3 respectively, can obtain sufficient amount of CCR6 with high homogeneity, strong cell function activity, stability and terminal differentiation effect phenotype⁺Th17 cells have an unexpected technical effect.

While the invention has been described in terms of one or more embodiments, it will be understood that the invention is not limited to those embodiments, and the description is intended to cover all alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims. All references cited herein are incorporated by reference in their entirety.

Claims

1. A method of differentiating and expanding Th17 cells in vitro, comprising the steps of:

(3) harvesting and detecting Th17 cells obtained in the step (2).

Wherein the cell culture medium in step (2) comprises a basal cell culture medium and additives comprising polarized cytokines (polarizing cytokines) and IL-2, wherein the polarized cytokines comprise one or more of TGF-beta, IL-1 beta, IL-6, IL-21, and IL-23, preferably the polarized cytokines are TGF-beta, IL-1 beta, IL-6, and IL-23; the concentration ratio of the IL-6 to the TGF-beta is 5-12.

2. The method according to claim 1, wherein the supplement in step (2) comprises TGF- β at a concentration of 1-10, preferably 2.5-10, more preferably 2.5ng/ml in the cell culture medium; the concentration of IL-6 in the cell culture medium is 10-50ng/ml, preferably 30 ng/ml.

3. The method according to claim 1, wherein the supplement in step (2) comprises IL-1 β at a concentration of 20ng/ml in cell culture medium, IL-23 at a concentration of 30ng/ml in cell culture medium, and IL-2 at a concentration of 10-30U/ml in cell culture medium.

4. The method of any one of claims 1-3, wherein the supplement of step (2) further comprises anti-CD 3 antibody and anti-CD 28 antibody, wherein the anti-CD 3 antibody and the anti-CD 28 antibody are soluble or in the form of immunomagnetic beads.

5. The method of claim 1, wherein the basal cell culture medium in step (2) can be serum-free cell culture medium or cell culture medium containing serum, the serum is 10-20% Fetal Bovine Serum (FBS), and the serum-free cell culture medium is selected from one or more of the following commercial culture media: yssel's T Cell Medium, Immunocult T Cell Medium, CTS OpTsizer T Cell expansion SFM and AIM V Medium, preferably Yssel's T Cell Medium, Immunocult T Cell Medium, more preferably Immunocult T Cell Medium.

6. The method of claim 1, wherein the cells of step (2) are cultured in the cell culture medium for at least 3 days, preferably 6 days, more preferably, the cells are isolated after the cells of step (1) are cultured in the cell culture medium for 3 days and the culture is continued to day 6 by adding fresh cell culture medium.

7. The method according to any one of claims 1 to 4, wherein the additive in step (2) further comprises an anti-IFN- γ antibody and an anti-IL-4 antibody.

8. The method of claim 1,the initial CD4 in the step (1)⁺Sources of T cells include, but are not limited to, human peripheral blood, cord blood, blood cells and blood precursor cells within immune organs (e.g., spleen or lymph nodes), the primary CD4⁺T cells include undifferentiated T cells expressing the surface antigen of CD4, including but not limited to human peripheral blood, cord blood, and other previously manipulated CD4⁺T cells. T cell precursors such as hematopoietic stem cells and lymphoblast cells obtained from bone marrow, umbilical cord blood, etc. can be induced to differentiate into CD4⁺T cells, the naive CD4⁺The T cells can be separated and purified from the monocyte population in the source by using a magnetic bead separation technology, and can also be commercialized separated and purified primary human CD4⁺CD45RA⁺T cells.

9. The method of claim 1, wherein the detecting in step (3) comprises surface markers or cell surface antigens of Th17 cells, including CD39, CD121a, CD161, CCR4, CCR6, CXCR3, IL-21R, and IL-23R, preferably CCR6 and CXCR 3; the transcription factor comprises ROR gamma t or ROR alpha; the secreted cytokines include IL-17A, IL-17AF, IL-17F, IL-21, and IL-22, preferably IL-17A.

10. A composition for selectively differentiating and expanding Th17 cells in vitro, the composition comprising polarized cytokines and IL-2, wherein the polarized cytokines comprise one or more of TGF- β, IL-1 β, IL-6, IL-21, and IL-23, preferably the polarized cytokines are TGF- β, IL-1 β, IL-6, and IL-23, the composition further comprising anti-CD 3 and anti-CD 28 antibodies and/or anti-IFN- γ, anti-IL-4 antibodies.

11. A method of producing Th17 cells using the composition of claim 9, the method comprising the steps of:

(1) acquisition of primary CD4+ T cells: the initial CD4⁺The T cell is CD4⁺CD45RA⁺A T cell;

(2) adding the composition into a basic cell culture medium to prepare the cell culture medium of the invention, and activating, differentiating and expanding the cells in the step (1) into Th17 cells in the cell culture medium, wherein the concentration of TGF-beta in the composition in the cell culture medium is 1-10ng/ml, preferably 2.5-10ng/ml, and more preferably 2.5 ng/ml; the concentration of IL-6 in the cell culture medium is 10-50ng/ml, preferably 30 ng/ml; the concentration ratio of the IL-6 to the TGF-beta is 5-12; the composition further comprises IL-1 β at a concentration of 20ng/ml in cell culture medium, IL-23 at a concentration of 30ng/ml in cell culture medium, and IL-2 at a concentration of 10-30U/ml in cell culture medium; the basic cell culture medium can be serum-free cell culture medium, and can also be cell culture medium containing 10-20% FBS;

(3) harvesting the Th17 cells obtained in the step (2) and detecting surface markers or cell surface antigens, transcription factors and secreted cytokines of the Th17 cells.

12. Use of the Th17 cell obtained by the method of claim 1 and/or the method of claim 11 for studying immunological mechanisms and/or detecting biological functions of drugs for treating diseases or disorders associated with Th17 cell dysregulation.

13. The use of claim 12, wherein the diseases or conditions associated with Th17 cell dysregulation include, but are not limited to, autoimmune diseases including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, psoriasis and other atopic and chronic inflammatory diseases.