Method for amplifying and inducing CD8 + T cells into CD8 + tissue settled T cells in vitro

Abstract

The invention provides a method for amplifying and inducing CD8 + T cells into CD8 + tissue settlement T cells in vitro, which comprises the following steps: (1) sorting mouse spleen CD8 + T cells by magnetic beads, and activating for 2 days under the stimulation of an anti-mouse CD3 monoclonal antibody and an anti-mouse CD28 monoclonal antibody, and (2) adding 10ng / mL TGF-beta and 20ng / mL IL-25 to induce the generation of the CD8 + tissue settlement T cells. According to the invention, IL-25 is innovatively used for in-vitro induction of CD8 + T cells into CD8 + tissue settlement T cells, the formation proportion of CD8 + tissue settlement T cells and expression of killer molecules are effectively improved, and the induction effect is better than that of single use of TGF-beta or combined use of TGF-beta and IL-15.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to a method for in vitro amplification and induction of CD8 + T cells are CD8 + Methods of tissue-resident T cells. Background Art

[0002] CD8 + Tissue resident memory T cells (TRM) are a unique group of cells that are distinct from circulating memory CD8 + The memory lymphocyte population of T cells mainly resides in peripheral non-lymphoid tissues, including barrier tissues such as skin, lungs, gastrointestinal tract, salivary glands and reproductive tract, and non-mucosal tissues such as liver, brain and pancreas. + T RM The cells share key phenotypic features, including downregulation of tissue egress-associated molecules and upregulation of tissue tethering molecules. CD69 is a membrane-bound type II C-lectin receptor that is a CD8 + T RM The key phenotypic characteristics of cells are to promote CD8 + T RM Cell residence. CD8 + T RM Another important feature of the cell is CD103, which is induced by TGF-β and can mediate tight adhesion to epithelial cells by binding to cadherin E expressed by epithelial cells, thereby promoting CD8 + T RM In addition to surface markers, CD8 + T RM Cell differentiation, survival and function require the coordinated action of multiple transcription factors, such as Blimp1, Hobit, Runx3 and BATF. + T RM Immune cells play an important role in local tissue defense against pathogen infection and tumors. They can directly kill target cells by secreting cytotoxic molecules (such as granzyme B and perforin), or they can indirectly play a protective role by secreting cytokines to activate or recruit other immune cells.

[0003] In recent years, T RM Cells have become a hot topic in the field of immunology, and more and more studies are focusing on CD8 + T RM Cells are being explored as therapeutic targets for tumors and inflammatory diseases. + TRM The proportion of cells is low and it is difficult to separate them from tissues. Therefore, it is difficult to establish an effective CD8 + T RM The cell differentiation system is particularly critical. Summary of the invention

[0004] The purpose of the present invention is to overcome the in vitro expansion and induction of CD8 + T RM Technical defects of cells, providing an in vitro expansion-induced CD8 + T cells are CD8 + Method for tissue resident T cells, method provided by the present invention and CD8 + T RM The cells are not only sufficient in number but also have strong cell functions; the method is simple and easy to operate; the CD8 + T RM Cells are suitable for primary Sjögren's syndrome syndrome, pSS), autoimmune skin diseases, multiple sclerosis (MS), and rheumatoid arthritis (RA) + T RM Research on the immunological mechanisms of diseases related to cell disorder.

[0005] The in vitro expansion-induced CD8 + The method by which T cells are tissue-resident T cells is as follows:

[0006] (1) Prepare mouse spleen single cell suspension and use magnetic beads to sort CD8 + T cells, the CD8 + T cells were placed in a 24-well culture plate coated with anti-mouse CD3 antibodies, and then anti-mouse CD28 antibodies and IL-2 were added to the culture plate and cultured for 48 hours to obtain A cell population;

[0007] (2) Collecting the A cell population in the culture plate in step (1), placing it in a new culture medium, adding IL-2, IL-25 and TGF-β, and continuing to culture for 48 hours to obtain a B cell population, and separating CD8 + T RM cell.

[0008] As a preferred embodiment of the present invention, the CD8 + The concentration of T cells was 2×10 6 / mL, and the culture medium volume was 1mL.

[0009] As a preferred embodiment of the present invention, the cell culture medium used in the culture plate in step (1) and the culture plate in step (2) is RPMI-1640 containing 10% FBS.

[0010] As a preferred embodiment of the present invention, the coating concentration of the anti-mouse CD3 antibody in step (1) is 1 μg / mL.

[0011] As a preferred embodiment of the present invention, the concentration of the anti-mouse CD28 antibody in step (1) is 1 μg / mL, and the concentration of the IL-2 in step (1) is 10 ng / mL.

[0012] As a preferred embodiment of the present invention, the concentration of the anti-mouse CD28 antibody in step (1) is 1 μg / mL, and the concentration of the IL-2 in step (1) is 10 ng / mL.

[0013] As a preferred embodiment of the present invention, the concentration of IL-2 in step (2) is 20 ng / mL.

[0014] As a preferred embodiment of the present invention, the CD8 + T RM Cells were tested, including the detection of the CD8 + T RM The proportion of cells in the B cell population and the detection of the CD8 + T RM Cell surface markers and secreted cytokines;

[0015] Wherein, the surface markers include CD8, CD69 and CD103;

[0016] The secreted cytokines include IFN-γ and granzyme B.

[0017] According to the technical solution of the present invention, the additive TGF-β can directly induce the expression of CD103 through Smad3, and can also indirectly upregulate the expression of CD103 by counteracting the inhibition of CD103 expression mediated by T-bet, Eomes and TCF1, thereby supporting CD103 + CD8 + The development and maintenance of TRM cells; at the same time, the presence of IL-2 is beneficial to CD8 + T cell survival.

[0018] Among them, interleukin-25 (IL-25) was first discovered in 2001. It is a member of the IL-17 cytokine family and is also known as IL-17E. However, IL-25 has low amino acid sequence homology with other members of the IL-17 family, so its biological effects are different from other members of the family. It is a unique cytokine in the IL-17 family. IL-25 was originally defined as a cytokine derived from Th2 cells. Later studies found that endothelial cells, epithelial cells, and CD8 + T cells, mast cells, dendritic cells (DC), macrophages, eosinophils and basophils, and group 2 innate lymphoid cells (ILC2) can all produce IL-25. The target cells of IL-25 include T cells, ILC2, myeloid cells, invariant natural killer T cells (iNKT), and some non-hematopoietic cells. Studies have found that after IL-25 binds to the receptor complex, it can stimulate cell proliferation, inhibit cell apoptosis, and induce the expression of various chemokines and adhesion molecules through a variety of signal transduction.

[0019] (1) The present invention innovatively adds IL-25 as an in vitro expansion inducing CD8 + Tissue-resident T cell inducers can stably and rapidly induce CD8 + T cell differentiation into CD8 + T RM cells, effectively increasing CD8 + T RM The induction effect of TGF-β alone or the combined use of TGF-β and IL-15 was better than that of TGF-β alone or the combined use of TGF-β and IL-15 in the formation ratio of cells and the expression of cytotoxic molecules IFN-γ and granzyme B.

[0020] (2) CD8 + T RM Cells are used to study immunological mechanisms and / or detect CD8 + T RM Among the biological functions of drugs for treating diseases or conditions related to cell disorder, CD8 + T RM Diseases related to cell disorder include but are not limited to autoimmune diseases (primary Sjögren's syndrome, autoimmune skin diseases, multiple sclerosis and rheumatoid arthritis) and other specific and chronic inflammatory diseases, laying the foundation for the study of their related biological functions and the immunological mechanisms of related diseases.

[0021] (3) CD8 + T RM Cells, whose transcriptome sequencing results can show that CD8 + T RM Changes in the expression of cell differentiation-related genes (S1pr1, Runx3, Sell, etc.).

[0022] (4) The method of the present invention is simple to operate, low in cost, and easy to promote and apply.

[0023] It should be appreciated that all combinations of the foregoing concepts, as well as additional concepts described in greater detail below, may be considered to be part of the inventive subject matter of the present disclosure, provided such concepts are not mutually inconsistent.

[0024] The foregoing and other aspects, embodiments and features of the present invention can be more fully understood from the following description in conjunction with the accompanying drawings. Other additional aspects of the present invention, such as the features and / or beneficial effects of the exemplary embodiments, will be apparent from the following description or learned from the practice of the specific embodiments according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings are not drawn to scale according to actual reference objects. In the accompanying drawings, each identical or nearly identical component shown in various figures may be represented by the same reference numeral. For the sake of clarity, not every component is labeled in each figure. Now, embodiments of various aspects of the present invention will be described by way of example and with reference to the accompanying drawings, in which:

[0026] Figure 1 In vitro culture of CD8 + T cells induced by IL-25 CD8 + T RM Expression of cell-related genes.

[0027] Figure 2 In vitro culture of CD8 + Expression of memory signature molecules by T cells induced by IL-25.

[0028] Figure 3 In vitro culture of CD8 + Expression of IFN-γ and granzyme B in T cells induced by IL-25. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be the common meanings understood by people with ordinary skills in the field to which the present invention belongs.

[0030] The words "first", "second" and similar words used in the patent application specification and claims of the present invention do not indicate any order, quantity or importance, but are only used to distinguish different components. Similarly, unless the context clearly indicates otherwise, the singular form of "a", "an" or "the" and similar words do not indicate a quantity limitation, but indicate the existence of at least one. Words such as "include" or "comprise" mean that the elements or objects appearing before "include" or "comprise" include the features, wholes, steps, operations, elements and / or components listed after "include" or "comprise", and do not exclude the existence or addition of one or more other features, wholes, steps, operations, elements, components and / or their collections. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0031] 1. Preliminary preparation

[0032] Antibody coating: dilute the anti-mouse CD3 antibody with sterile PBS buffer to a final concentration of 5 μg / μL, rinse the 24-well culture plate 2-3 times with sterile PBS buffer, add 200 μL of diluted anti-mouse CD3 antibody to the 24-well culture plate, and coat overnight at 4°C.

[0033] 2. Isolation and purification of mouse CD8 from mouse spleen + T cells

[0034] (1) Preparation of mouse spleen single cell suspension: 8-week-old C57BL / 6 mice were selected, killed and soaked in 75% ethanol solution for 5 min, spleens were removed on a sterile operating table, and the spleens were placed in a 70 μm mesh containing culture medium and fully ground, and the cell suspension was transferred to a sterile centrifuge tube, centrifuged at 4°C and 500g for 5 min, and the supernatant was discarded and the cells were flicked. 5 mL of sterile ACK was added, and the mixture was allowed to stand at room temperature for 5 min. An equal amount of RPMI-1640 culture medium containing 10% FBS was added to terminate the lysis, and the mixture was centrifuged at 4°C and 500g for 5 min. The supernatant was discarded and the cells were flicked. 10 mL of sterile PBE buffer was added, and the supernatant was discarded and the cells were flicked.

[0035] (2) Magnetic bead separation of CD8 + T cells: Take 100μL sterile PBE buffer to resuspend the cells, add 5μL / spleen CD8 magnetic beads, flick evenly, incubate at 4℃ for 30min, flick every 10min. Fix the magnetic column to the magnetic rack, rinse 3 times with 3mL sterile PBE buffer, then slowly add the cell suspension to the magnetic column. After the liquid drips off, add 3mL sterile PBE buffer to wash the column 3 times. Remove the magnetic column and place it in a 10mL centrifuge tube, add 5mL PBE, quickly push the column plug, repeat once, centrifuge at 500g for 5min, and the cell pellet obtained is CD8 + T cells.

[0036] 3. Induction of CD8 + T cell formation CD8 + T RM cell

[0037] (1) Inoculation of cells: sorted CD8 + T cells were resuspended in RPMI-1640 medium containing 10% FBS to obtain a concentration of 1×10 7 / mL cell suspension. Take the 24-well culture plate coated with anti-CD3 antibody the day before, aspirate the coating solution in the plate, and wash three times with 1mL sterile PBE buffer. Take 200μL of cell suspension and slowly inoculate it into the well, and add RPMI-1640 culture medium containing 10% FBS to make up the volume to 1mL. Add 1μg / mL anti-mouse CD28 antibody and 10ng / mL IL-2 to each well. Place the culture plate in a 37℃ 5% CO2 incubator for 48h to obtain A cell population.

[0038] (2) Replacement of medium: Collect cell population A in the culture plate, centrifuge at 500 g for 5 min at 4°C, discard the supernatant, flick the cells to mix, add 10 mL of sterile PBS buffer and wash once, adjusting the cell concentration to 1 × 10 7 / mL, inoculated in a new 24-well culture plate. Set up three groups and mark them: the first group is a blank group, adding IL-2 at a concentration of 20ng / mL; the second group is a control group, adding IL-2 at a concentration of 10ng / mL and TGF-β at a concentration of 10ng / mL; the third group is an experimental group, adding IL-2 at a concentration of 10ng / mL and TGF-β at a concentration of 10ng / mL. The above three groups of embodiments are respectively added with IL-25 at different concentrations (respectively 10ng / mL, 20ng / mL, and 50ng / mL). The culture plate was placed in a 37℃ 5% CO2 incubator and cultured for 48h to obtain three different B cell populations, namely B1, B2, and B3.

[0039] In addition, we compared the effects of IL-15 and IL-25 on CD8 + T RM The induction effect of in vitro expansion, the groups are as follows: the first group is a blank group, adding IL-2 at a concentration of 20ng / mL; the second group is a control group, adding IL-2 at a concentration of 10ng / mL, TGF-β at 10ng / mL and IL-15 at 20ng / mL; the third group is an experimental group, adding IL-2 at a concentration of 10ng / mL, TGF-β at 10ng / mL and IL-15 at 20ng / mL, and the above three groups of experimental examples are added with different concentrations (10ng / mL, 20ng / mL, 50ng / mL) of IL-25. The culture plate was placed in a 37℃ 5% CO2 incubator and cultured for 48h to obtain three different groups of C cell populations, namely C1, C2, and C3.

[0040] 4. Detection and analysis

[0041] On day 4, the induced cells were analyzed by flow cytometry.

[0042] (1) Surface staining to detect CD8 + CD69 + CD103 + T RM The proportion of cells

[0043] Collect cells (B cell population or C cell population) in the culture plate into EP tubes, centrifuge at 4℃500g for 5min, discard the supernatant and flick the cells, add 1mL PBS buffer to wash once, centrifuge at 4℃500g for 5min, discard the supernatant and flick the cells, add 100μL PBS buffer to resuspend the cells, add 0.5μL of surface antibodies each, and incubate at 4℃ in the dark for 30min; add 1mL PBS buffer to wash once, centrifuge at 4℃500g for 5min, discard the supernatant and flick the cells, add 200μL PBS buffer to resuspend the cells, add 40μL paraformaldehyde solution to fix, and store in the dark.

[0044] RNA-seq results showed that TGF-β-induced CD8 + T RM Compared with the cells, the addition of IL-25 induced the production of CD8 + T RM The cells expressed lower levels of tissue export molecules (such as S1pr1 and Sell) and higher levels of tissue tethering molecules (Itgae and Cd69) and functional molecules (Fasl and Gzmb).

[0045] Flow cytometry detection of CD8 + CD69 + CD103 + T RM The results of cell ratio are as follows Figure 2 As shown, in the traditional CD8 + T RM The cell induction system (TGF-β+IL-15) or the addition of IL-25 (10ng / mL, 20ng / mL, 50ng / mL) to TGF-β can significantly promote CD8 + T RM Cell differentiation, among which 20ng / mL IL-25 had the most obvious promoting effect.

[0046] (2) Detection of CD8 by membrane permeabilization staining + T RM Function of IFN-γ and granzyme B secretion by cells

[0047] The collected cells (B cell population or C cell population) were seeded into a new 24-well culture plate, and the culture medium was added to 1 mL of liquid in the well, and 5 μg / mL PMA, 4 μg / mL Monensin and 1 μg / mL Ionomycin were added, and the cells were incubated in a 37°C 5% CO2 incubator for 1 hour. After the incubation, the cells in the culture plate were collected into an EP tube, centrifuged at 4°C 500g for 5 minutes, and the supernatant was discarded and 100 μL PBS buffer and 100 μL Intracelluar Fix & Perm Buffer were added, mixed, and incubated at 4°C for 20 minutes. 1 mL of washing solution (Permeabilization Buffer: ddH2O = 1:9) was added to terminate the membrane permeabilization, centrifuged at 4°C 500g for 5 minutes, and the supernatant was discarded and 40 μL of washing solution was added to resuspend, and 1 μL of intracellular antibody was added, and incubated at 4°C in the dark for 45 minutes. Add 1 mL of washing solution, centrifuge at 500 g for 5 min at 4°C, discard the supernatant, add 200 μL of PBS buffer to resuspend the cells, add 40 μL of paraformaldehyde solution to fix, and store in the dark.

[0048] Flow cytometry detection of CD8 + T RMThe results of IFN-γ and granzyme B secretion levels were as follows Figure 3 As shown, CD8 + T RM The cell proportion was significantly increased, and the expression of granzyme B was also higher.

[0049] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. A person with ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the definition of the claims.

Claims

1. An in vitro expansion-induced CD8 + T cells are CD8 + Tissue-resident T cells It is characterized in that The following steps are involved: (1) Prepare mouse spleen single cell suspension and use magnetic beads to sort CD8 + T cells, the CD8 + T cells were placed in a 24-well culture plate coated with anti-mouse CD3 antibodies, and then anti-mouse CD28 antibodies and IL-2 were added to the culture plate and cultured for 48 hours to obtain A cell population; (2) collecting the A cell population in the culture plate in step (1), placing it in a new culture plate, adding IL-2, IL-25 and TGF-β, and continuing to culture for 48 hours to obtain a B cell population, and separating CD8 + T RM cell.

2. The method according to claim 1, It is characterized in that CD8 + The concentration of T cells was 2×10 6 / mL, and the culture medium volume was 1mL.

3. The method according to claim 1, It is characterized in that The cell culture medium used in the culture plate of step (1) and the culture plate of step (2) is RPMI-1640 containing 10% FBS.

4. The method according to claim 1, It is characterized in that The coating concentration of the anti-mouse CD3 antibody in step (1) is 1 μg / mL.

5. The method according to claim 1, It is characterized in that The concentration of the anti-mouse CD28 antibody in step (1) is 1 μg / mL, and the concentration of the IL-2 in step (1) is 10 ng / mL.

6. The method according to claim 1, It is characterized in that The concentration of IL-2 in step (2) is 20 ng / mL.

7. The method according to claim 1, It is characterized in that The concentration of the TGF-β in the step (2) is 10 ng / mL.

8. The method according to claim 1, It is characterized in that The concentration of the IL-25 in the step (2) is 10 ng / mL-50 ng / mL.

9. The method according to claim 8, It is characterized in that The concentration of IL-25 in step (2) is 20 ng / mL.

10. The method according to claim 1, It is characterized in that The CD8 + T RM Cells were tested, including the detection of the CD8 + T RM The proportion of cells in the B cell population, and Detection of the CD8 + T RM Cell surface markers and secreted cytokines; Wherein, the surface markers include CD8, CD69 and CD103; The secreted cytokines include IFN-γ and granzyme B.

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