A method of in vitro induction of tissue-resident memory CD8+ T cells

Abstract

The application discloses a method for inducing tissue-resident memory CD8+ T cells in vitro and belongs to the technical field of cell culture. + CD69 + CD103 + The proportion of TRM can be up to 70%, and CD8 + CD69 + CD103 + The TRM phenotype is CD122 + , and expresses CD44 and CD62L which are characteristic molecules of central memory T cells. Meanwhile, the method uses macrophages as inducers, and can provide the possibility of inducing antigen-specific TRM.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell culture, and particularly relates to a method for inducing tissue-resident memory CD8+ T cells in vitro. BACKGROUND

[0002] Resident memory T cells (TRM) are a kind of more completely differentiated memory T cells expressing CD69 or CD103 or CD49a, which are different from traditional central memory T cells (TCM) and effector memory T cells (TEM). TRM does not circulate and exists in tissues. TRM can rapidly initiate secondary immunity and is the first T cell to respond to antigen recognition or inflammation. TRM is rapidly activated and differentiated into TEM and TCM when stimulated, and plays a key role in mediating peripheral tissue adaptive immune response. It is previously believed that TRM mainly exists in epithelial barrier tissues, including gastrointestinal tract, lung, skin and reproductive tract. In recent years, researches have shown that TRM also exists in non-barrier tissues, including kidney, liver, brain, pancreatic islet and synovial tissue. Therefore, the activation of TRM function is involved in the regulation of the progression and prognosis of multiple system diseases, and plays an important role in skin-related diseases, various types of solid tumors and chronic kidney disease.

[0003] The method for inducing and culturing TRM in vitro is an important method for studying the function of TRM. Some recent literatures have reported that IL-15 and TGF-β cross-stimulation can be used to induce TRM (You Z, Li Y. Nature communications. 2021; 74(2): 847-63). In this method, human PBMCs are separated, and IL-15 (50 ng / ml) is added to the culture medium 3 days before the experiment. The culture medium is replaced on the third day of the experiment, and then TGF-β (50 ng / ml) is added for continuous culture for 3 days. We found that the CD8 + CD69 + CD103 + TRM accounts for only about 15% of CD8+ T cells, and the induction efficiency is low. Moreover, this research only attempts to use human PMBC samples. SUMMARY

[0004] To solve the above problems, the primary purpose of the present application is to provide an application of macrophages (BMDMs) in inducing tissue-resident memory T cells in vitro.

[0005] Another purpose of the present application is to provide a method for inducing tissue-resident memory CD8 + T cells in vitro.

[0006] In order to achieve the above-mentioned object, the present application adopts the following technical solutions.

[0007] Application of macrophages in inducing tissue-resident memory T cells in vitro.

[0008] Further, the application is to induce CD8 + T cells to generate tissue-resident memory CD8 + T cells in vitro.

[0009] A method for inducing tissue-resident memory CD8 + T cells in vitro, comprising the following steps:

[0010] S1, obtaining macrophages;

[0011] S2, obtaining CD8 + naive T cells;

[0012] S3, co-culturing the macrophages and the CD8 + naive T cells.

[0013] Further, the tissue-resident memory CD8 + T cells have higher expression of CD122, CD44 and / or CD62 on the surface. + CD69 + CD103 + T cells.

[0014] Further, the tissue-resident memory CD8 + T cells have higher expression of CD122, CD44 and / or CD62 on the surface.

[0015] Further, in the step S1, the macrophages are any one or a combination of M0-like macrophages, M1-like macrophages, M-IL4-like macrophages and M-IL10-like macrophages. The M-IL4-like macrophages and M(IL4)-like macrophages in the present application refer to macrophages induced by IL4, and the M-IL10-like macrophages and M(IL10)-like macrophages refer to macrophages induced by IL10.

[0016] Further, in the step S1, the specific operation for obtaining the macrophages is as follows:

[0017] ①Bone marrow-derived macrophages are resuspended in a culture medium containing M-CSF (macrophage colony-stimulating factor) after being lysed by red blood cells, and are cultured, and after 6±1 days of induction, the culture medium without M-CSF is replaced, and the macrophages are rested for 24±2h to obtain M0-like macrophages; or,

[0018] ②On the basis of operation ①, the obtained M0-like macrophages are replaced into a culture medium containing LPS (lipopolysaccharide) and IFN-Γ, and cultured for 12±2h to obtain M1-like macrophages; or,

[0019] ③On the basis of operation ①, the obtained M0-like macrophages are replaced into a culture medium containing IL-4, and cultured for 12±2h to obtain M-IL4-like macrophages; or,

[0020] ④On the basis of operation ①, the obtained M0-like macrophages are replaced into a culture medium containing IL-10, and cultured for 12±2h to obtain M-IL10-like macrophages.

[0021] Further, in the culture medium containing M-CSF, the concentration of M-CSF is 50±2ng / ml; in the culture medium containing LPS and IFN-Γ, the concentration of LPS is 50±5ng / ml, and the concentration of IFN-Γ is 20±2ng / ml; in the culture medium containing IL-4, the concentration of IL-4 is 20±2ng / ml; and in the culture medium containing IL-10, the concentration of IL-10 is 20±2ng / ml.

[0022] Further, the concentration of the resuspended cells is adjusted to 2×10 6 cells / ml.

[0023] Further, the resuspended cells are cultured in a 37℃ incubator, and the medium is replaced every 2-3 days.

[0024] Further, the culture medium refers to 1640 complete culture medium.

[0025] Further, in step S2, the CD8 + initial T cells are CD8 + CD122 - initial T cells.

[0026] Further, in step S2, the CD8 + initial T cells are obtained by the following specific operation: the spleen is aseptically removed, ground to prepare a single cell suspension, and the single spleen cells are obtained by red blood cell lysis, and then CD8, CD122 antibodies are added for incubation, and CD8 + CD122 - initial T cells are sorted.

[0027] Further, in step S3, the macrophages and CD8 + initial T cells are co-cultured by the following specific operation: after the macrophages are cultured to adhere to the cells, CD8 +After the initial T cell co-culture, the suspension T cells were collected after 4±0.5 days of continuous culture for phenotype analysis to obtain CD8 + CD69 + CD103 + tissue-resident memory T cells.

[0028] Further, in step S3, the concentration of macrophages in the culture system is 1×10 5 macrophages per well, and the concentration of CD8 + initial T cells is 2.0×10 5 macrophages per well.

[0029] The present application has the following advantages and effects over the prior art:

[0030] There is no method for inducing mouse TRM in vitro at present, and the only literature about the in vitro culture of TRM is to stimulate human PBMCs with a combination of cytokines to induce human TRM. The present application uses macrophages as inducers to induce TRM by co-culturing with T cells.

[0031] The present application uses mouse primary macrophages to co-culture with spleen T cells to induce TRM, and the T cells are collected for phenotype analysis at 4 days of co-culture. + CD69 + CD103 + The proportion of TRM can be as high as 70%, and the CD8 + CD69 + CD103 + TRM has the phenotype of CD122 + and expresses the CD44 and CD62L characteristic molecules of central memory T cells. Meanwhile, the present method uses macrophages as inducers, which can provide the possibility of inducing antigen-specific TRM.

[0032] The present application successfully induces CD8 + CD69 + CD103 + TRM by using macrophages for the first time, and the efficiency is higher than that of the existing method of stimulating PBMCs with a combination of IL-15 / TGF-β to induce TRM. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 are analysis diagrams of the effect of co-culturing different types of BMDMs with CD8 + CD122 - T cells to induce TRM; wherein A and D are TRM (CD8 + CD69 + CD103+ )Flow analysis chart and statistical analysis results, B and E are CD8 + CD122 + Flow analysis chart and statistical results of expression, C and F are CD44 + CD62L + Expression analysis. DETAILED DESCRIPTION

[0034] The present application will be further described in conjunction with the examples and drawings, but the embodiments of the present application are not limited thereto.

[0035] Example 1

[0036] (1) Preparation of bone marrow-derived macrophages: 6-8-week-old female C57BL / 6 mice were selected, the tibia and femur were aseptically stripped, and the bone marrow cavity was repeatedly washed with 1.0 ml syringe containing 5% double-antigen 1640 culture medium to obtain bone marrow cells, which were passed through a 200-mesh nylon mesh to obtain a single-cell suspension of bone marrow cells, which were lysed with red blood cells and resuspended with 1640 complete culture medium containing M-CSF (50 ng / ml), and the cell concentration was adjusted to 2×10 6 / ml, and cultured in a 37℃ incubator, the medium was changed every 2-3 days, and after 6 days of induction, the medium was replaced with 1640 complete culture medium without M-CSF, and the macrophages were rested for 24 h, at which time the macrophages were M0-like macrophages, and the medium was replaced with 1640 medium containing LPS (5.0 ng / ml) and IFN-Γ (20 ng / ml) for 12 h to induce M1-like BMDMs, or IL-4 (20 ng / ml) was added for 12 h to induce M(IL4) macrophages, or IL-10 (20 ng / ml) was added for 12 h to induce M(IL10) macrophages.

[0037] (3) Sorting Cells: 6-8-week-old female C57BL / 6 mice were selected, the spleen was aseptically removed, and a single-cell suspension was prepared by grinding, and single spleen cells were obtained by red blood cell lysis, and anti-mouse CD8, CD122 antibodies were added for incubation, and CD8 + CD122 - naive T cells, the sorted naive T cells were counted, and resuspended with 1640 complete culture medium to a cell density of 2.0×10 6 cells / ml.

[0038] (4) Macrophage / T cell co-culture: M0-like, M1-like, M(IL4)-like or M(IL10)-like macrophages were added to a 96-well plate at 1×10 5 cells / well, 100 μl of medium / well, and after the cells adhered, 2.0×10 T cells / well were added to each well.6 T cells were co-cultured (cells / ml) for 4 consecutive days. Suspension T cells were then collected and incubated with anti-mouse CD8, CD122, CD69, CD103, CD44, and CD62L. Flow cytometry was used to detect TRM differentiation and phenotype. Results are as follows: Figure 1 As shown, CD8 was successfully induced. + CD69 + CD103 + Tissue-resident memory T cells, accounting for up to 70%, and CD8 + CD69 + CD103 + The TRM phenotype is CD122. + CD122 is the βc chain of the IL-15 receptor, suggesting that TRM cells induced by this method have a higher response to IL-15, and that the TRM cells induced by this method exhibit characteristics of central memory T cells (CD44). + CD62L + ).

[0039] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method of in vitro inducing tissue-resident memory CD8 + T cells, characterized in that: The method comprises the following steps: S1, obtaining bone marrow-derived macrophages; S2, obtaining spleen-derived CD8 + naive T cells; S3, macrophages and CD8 + Initial T cell co-culture; In the step S1, the macrophages are any one of M0-like macrophages, M1-like macrophages, M-IL4-like macrophages and M-IL10-like macrophages; In step S2, the CD8 + Naive T cells are CD8 + CD122 - Naive T cells; The step S3 is to co-culture the macrophages and CD8 + The specific operation of the initial T cell co-culture is as follows: after the macrophages are cultured to adhere to the cells, the CD8 + The specific operation of the initial T cell co-culture is as follows: after the macrophages are cultured to adhere to the cells, the CD8 + CD69 + CD103 + The tissue-resident memory T cell is obtained by co-culturing the initial T cells with the macrophages in the culture system, and the concentration of the macrophages is 1×10 5 CD8 + The concentration of the initial T cells is 2.0×10 5 CD8 + The concentration of the initial T cells is 2.0×10 + CD69 + CD103 + T cell.

2. The method of in-vitro inducing tissue-resident memory CD8 + T cells according to claim 1, characterized in that: The tissue-resident memory CD8 + T cells have higher expression of CD122, CD44, and / or CD62 on their surface.

3. The method of in vitro inducing tissue-resident memory CD8 + T cells according to claim 1, characterized in that: In the step S1, the specific operation for obtaining the macrophages is as follows: (1) resuspending the bone marrow-derived macrophages after red blood cell lysis with a culture medium containing M-CSF, culturing, replacing the culture medium without M-CSF after 6±1 days of induction, and resting for 24±2 h to obtain M0-like macrophages; or, (2) on the basis of the operation (1), replacing the obtained M0-like macrophages into a culture medium containing LPS and IFN-γ, culturing for 12±2 h to obtain M1-like macrophages; or, (3) on the basis of the operation (1), replacing the obtained M0-like macrophages into a culture medium containing IL-4, culturing for 12±2 h to obtain M-IL4-like macrophages; or, (4) on the basis of the operation (1), replacing the obtained M0-like macrophages into a culture medium containing IL-10, culturing for 12±2 h to obtain M-IL10-like macrophages; In the culture medium containing M-CSF, the concentration of M-CSF is 50±2 ng / ml; in the culture medium containing LPS and IFN-γ, the concentration of LPS is 50±5 ng / ml and the concentration of IFN-γ is 20±2 ng / ml; in the culture medium containing IL-4, the concentration of IL-4 is 20±2 ng / ml; and in the culture medium containing IL-10, the concentration of IL-10 is 20±2 ng / ml.

4. The method of in vitro inducing tissue-resident memory CD8 + T cells according to claim 1, characterized in that: The step S2 obtains CD8 + The specific operation of the initial T cell is as follows: the spleen is taken out aseptically, grinded to prepare a single cell suspension, and the single spleen cell is obtained by red blood cell lysis, CD8 and CD122 antibodies are added for incubation, and CD8 + CD122 - The initial T cell.

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