Preparation method and application of double-negative T cell

Abstract

The invention relates to the technical field of biological medicine, in particular to a preparation method and application of double-negative T (DNT) cells. The invention provides a preparation method of double-negative T cells, which comprises the following steps: separating mononuclear cells in peripheral blood or lymphatic tissues to obtain a mononuclear cell population; and removing the CD4T cell, the CD8T cell, the NK cell, the granulocyte, the mononuclear macrophage, the gamma-delta T cell and the B cell in the mononuclear cell population to obtain the double-negative T cell. According to the preparation method provided by the invention, in-vitro amplification culture and removal of cells secreting IL-17 are not needed, the cells are directly purified and then injected to the local part of the tissue, and the high-proportion TCR alpha beta + DNT cell level is achieved locally, so that the effect of preventing and treating autoimmune diseases and inflammatory diseases is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a method for preparing double-negative T cells and its applications. Background Art

[0002] TCRαβ+ double-negative T cells (DNT cells) are a type of T lymphocytes that do not express CD4 and CD8 molecules, nor do they express surface markers of NK cells, i.e., human CD3+TCRαβ+CD4-CD8-CD56- (mouse CD3+TCRαβ+CD4-CD8-NK1.1-), and play multiple important roles in maintaining normal immunity of the body. TCRαβ+ DNT cells have a direct killing and inhibitory effect on inflammatory immune cells and tumor cells, and have good preventive and therapeutic effects on various autoimmune diseases, organ transplantation rejection, GVHD, inflammation-related diseases, and tumors, and can be used as a potential cell therapy technology for the prevention and treatment of autoimmune diseases, inflammation-related diseases, and tumors.

[0003] Existing TCRαβ+ DNT cell therapy research mainly involves intravenous infusion, using the chemotactic effect of DNT cells to reach the lesion site for disease treatment. This requires DNT cells to be amplified and cultured in vitro to reach a certain quantity level before they can play a therapeutic role. For example, the preparation methods provided by the invention patents with publication numbers CN101313061B or CN106191062B both require in vitro amplification and culture of DNT cells to reach the cell quantity for treatment. In vitro amplification and culture not only easily cause cell contamination, but also the components or states of cells may change after cell culture. After in vitro amplification and culture, the cells need to be subjected to various detections to exclude contamination, cell trait changes, etc., resulting in high usage costs. Moreover, the cells after in vitro amplification and culture need to be cryopreserved and can only be used for treatment after the test items are normal. Cryopreservation will also have an irreversible impact on the quality of the cells. Summary of the Invention

[0004] To solve the above problems, the present invention provides a method for preparing double-negative T cells and its applications. The double-negative T cells prepared by the preparation method of the present invention do not require cell culture, and can be directly purified and injected into the local tissue, without generating the above-mentioned drawbacks of long-term cell culture.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing double-negative T cells, comprising the following steps:

[0007] Isolate mononuclear cells from peripheral blood or lymphoid tissue to obtain a mononuclear cell population;

[0008] Remove CD4 T cells, CD8 T cells, NK cells, granulocytes, mononuclear macrophages, gamma-delta T cells, and B cells from the single nuclear cell population to obtain double-negative T cells.

[0009] Preferably, the reagent used for separation includes lymphocyte separation medium.

[0010] Preferably, the method for removal includes immunomagnetic bead method.

[0011] Preferably, the antibodies used in the immunomagnetic bead method include: CD4 antibody, CD8α antibody, CD8β antibody, NK antibody, CD11b antibody, CD14 antibody, TCRγδ antibody, and CD19 antibody; the NK antibody is NK1.1 or CD56.

[0012] Preferably, the antibody is a PE-labeled antibody, FITC-labeled antibody, or Biotin-labeled antibody.

[0013] Preferably, the lymphoid tissue includes spleen or lymph node.

[0014] The present invention provides the use of double-negative T cells prepared by the preparation method described in the above technical solution in the preparation of products for preventing and treating autoimmune diseases and / or inflammatory diseases.

[0015] Preferably, the inflammatory disease includes one or more of periodontitis, arthritis, spinal cord injury, and spinal cord inflammation.

[0016] The present invention provides a preparation for preventing and treating periodontitis, and the active ingredient of the preparation includes double-negative T cells; the double-negative T cells are double-negative T cells prepared by the preparation method described in the above technical solution.

[0017] Preferably, the dosage form of the preparation includes an injection; the solvent of the injection includes physiological saline.

[0018] Beneficial effects:

[0019] The present invention provides a method for preparing double-negative T cells, comprising the following steps: isolating mononuclear cells from peripheral blood or lymphoid tissue to obtain a mononuclear cell population; removing CD4 T cells, CD8 T cells, NK cells, granulocytes, mononuclear macrophages, gamma-delta T cells, and B cells from the mononuclear cell population to obtain double-negative T cells. The preparation method provided by the present invention obtains double-negative T cells (DNT) that can inhibit inflammation by removing cells that promote local inflammatory responses, and can be used to prepare products for preventing and treating autoimmune diseases and / or inflammatory diseases. The method provided by the present invention does not require in vitro amplification culture and removal of cells secreting IL-17, and can be directly purified and injected into the local tissue, so that a relatively high proportion of TCRαβ+DNT cell level can be achieved locally, thereby achieving the effect of preventing and treating autoimmune diseases and inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0021] Figure 1 It is a typical flow cytometry detection diagram of TCRαβ+DNT cells in human periodontal tissue; among them, the left figure is normal human periodontal tissue, and the right figure is diseased periodontal tissue of periodontitis patients;

[0022] Figure 2 It is the statistical analysis result of the percentage of TCRαβ+DNT cells in total T cells in the periodontal tissues of normal people (HC) and periodontitis patients (CP);

[0023] Figure 3 It is a typical flow cytometry detection diagram of TCRαβ+DNT cells in mouse periodontal tissue;

[0024] Figure 4 It is the statistical analysis result of the percentage of TCRαβ+DNT cells in total T cells in the periodontal tissues of normal mice (left side) and periodontitis mice (right side);

[0025] Figure 5 The upper figure is a typical CT imaging diagram of periodontal tissue; the lower figure is a typical hematoxylin-eosin staining diagram of periodontal tissue;

[0026] Figure 6 It is a statistical analysis diagram of the alveolar bone resorption index (CEJ-ABC distance) of mice;

[0027] Figure 7 It is the comparison result of the expression levels of inflammatory factor mRNAs in periodontal tissue;

[0028] Figure 8 It is a typical CT imaging diagram of periodontal tissue;

[0029] Figure 9 Statistical analysis chart of the alveolar bone resorption index (CEJ-ABC distance) in mice;

[0030] Figure 10 Typical images of immunofluorescence staining of neutrophils (Ly6G) and IL-1β in periodontal tissues; scale bar: 100 μm;

[0031] Figure 11 Statistical analysis results of the absolute count of neutrophils (Ly6G) in periodontal tissues;

[0032] Figure 12 Statistical analysis results of the proportion of neutrophils (Ly6G) secreting IL-1β in periodontal tissues;

[0033] Figure 13 Typical images of immunofluorescence staining of neutrophils (Ly6G) and extracellular trap marker citrullinated histone H3 (Cit-H3) in periodontal tissues; scale bar: 100 μm;

[0034] Figure 14 Statistical analysis results of the proportion of neutrophils (Ly6G) secreting extracellular traps (Cit-H3) in periodontal tissues;

[0035] Among them, * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. Detailed implementation manners

[0036] The present invention provides a method for preparing double-negative T cells, comprising the following steps:

[0037] Isolate mononuclear cells from peripheral blood or lymphoid tissues to obtain a mononuclear cell population;

[0038] Remove CD4 T cells, CD8 T cells, NK cells, granulocytes, mononuclear macrophages, gamma-delta T cells, and B cells from the mononuclear cell population to obtain double-negative T cells.

[0039] The TCRαβ+DNT cells prepared by the present invention do not need to be amplified and cultured in vitro and can be directly used for local injection of autologous tissues, achieving a relatively high proportion of TCRαβ+DNT cell levels locally, thereby playing a role in preventing and treating autoimmune diseases and inflammatory diseases.

[0040] As an implementation manner, the reagent used for isolation can be lymphocyte separation medium.

[0041] As an implementation manner, the separation method includes: adding lymphocyte separation solution with the same volume as the blood into a centrifuge tube (labeled A); simultaneously, adding PBS with the same volume as the blood into another centrifuge tube (labeled B); transferring the peripheral blood into centrifuge tube B, gently mixing to obtain a PBS mixture; moderately tilting centrifuge tube A, and slowly adding the PBS mixture along the wall; centrifuging at a speed of 400g for 20 minutes at room temperature; taking out the centrifuge tube after centrifugation, and sucking the lymphocyte layer therein to obtain a mononuclear cell population (PBMC).

[0042] As an implementation manner, the removal method can be the immunomagnetic bead method.

[0043] As an implementation manner, the antibodies used in the immunomagnetic bead method include: CD4 antibody, CD8α antibody, CD8β antibody, NK antibody, CD11b antibody, CD14 antibody, TCRγδ antibody, and CD19 antibody; the NK antibody is NK1.1 or CD56.

[0044] As an implementation manner, the antibody is a PE-labeled antibody, a FITC-labeled antibody, or a Biotin-labeled antibody. As another implementation manner, the antibody is a PE-labeled antibody.

[0045] As an implementation manner, the lymphoid tissue can be the spleen or lymph nodes.

[0046] The present invention provides the application of the double-negative T cells prepared by the preparation method described in the above technical solution in the preparation of products for preventing and treating autoimmune diseases and / or inflammatory diseases.

[0047] As an implementation manner, the inflammatory diseases include one or more of periodontitis, arthritis, spinal cord injury, and spinal cord inflammation. As another implementation manner, the inflammatory disease is periodontitis. As an implementation manner, the product can be a drug. As an implementation manner, the inflammatory diseases include diseases caused by one or more of inflammatory factors IL-1β, TNFα, CCL2, and IL-17A.

[0048] The present invention provides a preparation for preventing and treating periodontitis, and the active ingredient of the preparation includes double-negative T cells; the double-negative T cells are double-negative T cells prepared by the preparation method described in the above technical solution.

[0049] As an implementation manner, the dosage form of the preparation can be an injection; the solvent of the injection can be physiological saline. As an implementation manner, based on each mouse, the unit effective dose of the preparation can be 1000 double-negative T cells.

[0050] To further illustrate the present invention, a method for preparing a double-negative T cell and its application provided by the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] Example 1

[0052] Peripheral blood PBMCs (mononuclear cells) were isolated using lymphocyte separation medium (Ficoll), and then CD4 T cells, CD8 T cells, NK cells, granulocytes, mononuclear macrophages, gamma-delta T cells, and B cells were removed by CD4, CD8α antibody, CD8β antibody, NK antibody, CD11b, CD14, TCRγδ, and CD19 antibody labeling + magnetic bead separation. The unlabeled cells obtained were DNT cells, and the NK antibody was NK1.1 (mouse) / CD56 (human); the specific steps are as follows:

[0053] Collect a blood sample in an EDTA anticoagulant tube. Add lymphocyte separation medium equal in volume to the blood to a 50 mL centrifuge tube (labeled A). At the same time, add PBS equal in volume to the blood to another 50 mL centrifuge tube (labeled B). Transfer the blood sample to centrifuge tube B and gently mix to obtain a PBS mixture. Tilt centrifuge tube A moderately and slowly add the PBS mixture along the wall. Centrifuge at a speed of 400 g for 20 minutes at room temperature. After centrifugation, take out the centrifuge tube and aspirate the PBMCs therein. The washed PBMCs were incubated with PE-labeled CD4, CD8α, NK antibody, CD11b, CD14, TCRγδ, and CD19 antibodies for 30 minutes, washed, and then incubated with anti-PE magnetic beads for 30 minutes and washed again. After removing the cells labeled with magnetic beads using a magnetic field, the unlabeled cells retained by negative selection were DNT cells.

[0054] Example 2

[0055] Obtain gingival tissues from healthy periodontal sites and gingival tissues from sites with severe chronic periodontitis. The diagnosis of periodontitis is based on comprehensive clinical examinations, probing depth measurements, clinical attachment loss assessments, and confirmation by alveolar bone X-ray films. Single-cell suspensions of gingival tissues were obtained by mechanical chopping and collagenase IV digestion. Fluorescent antibodies were used to stain and label cell surface markers CD45, CD3, CD4, CD8, TCRαβ, and CD56. Flow cytometry was used to detect the changes in the proportion of immunocytes TCRαβ+CD4-CD8-CD56-T (TCRαβ+DNT) cells. The results are shown in Figure 1 and Figure 2 .

[0056] In normal human peripheral lymphoid tissues (such as spleen, lymph nodes, etc.), TCRαβ+ DNT cells only account for 1-3% of the total number of T cells. Compared with peripheral lymphoid tissues, normal human periodontal tissues have a higher proportion of TCRαβ+ DNT cells, accounting for 14.42±2.2% of the total number of T cells in periodontal tissues. However, the proportion of TCRαβ+ DNT cells in chronic periodontitis tissues decreases, only accounting for 9.6±2.35% ( Figure 1 and Figure 2 ).

[0057] Example 3

[0058] For C57BL / 6 mice, a periodontitis model was established by ligating the maxillary second molar with 4-0 silk thread. Seven days after modeling, gingival tissues were obtained. The gingival tissues were mechanically minced and digested with collagenase IV to obtain a single-cell suspension of gingival tissues. Fluorescent antibodies were used to stain and label cell surface markers CD45, CD3, CD4, CD8, TCRαβ, and NK. Flow cytometry was used to detect the change in the proportion of TCRαβ+CD4-CD8-NK1.1-T cells, an immune cell. The results are shown in Figure 3 and Figure 4 .

[0059] The results showed that, consistent with the detection results of human periodontal tissues, normal C57BL / 6 mouse periodontal tissues had a higher proportion of TCRαβ+ DNT cells (18.86±1.3%), while the proportion of TCRαβ+ DNT cells in periodontitis model mouse periodontal tissues decreased (17.08±1.12%) ( Figure 3 and Figure 4 ).

[0060] Example 4

[0061] Mouse spleens and lymph nodes were isolated. After grinding and lysing red blood cells, mononuclear cells were obtained. PE-labeled antibodies (CD4, CD8α, CD8β, NK1.1, CD14, CD11b, TCRγδ, and CD19) were used to label cell surface markers. Anti-PE magnetic beads (Anti-PE Microbeads, purchased from Miltenyi Biotec GmbH, Germany, catalog number: 130-048-801) were used for sorting to obtain TCRαβ DNT cells (the method was referred to Example 1), and verification was performed by flow cytometry (purity >98%) to obtain DNT cells.

[0062] Normal saline and the DNT cells were mixed to prepare DNT cell suspensions with concentrations of 1000 cells / 5 μl and 5000 cells / 5 μl, denoted as DNT Low and DNT High, respectively.

[0063] C57BL / 6 mice were used to establish a periodontitis model by ligating the maxillary second molar with 4-0 silk thread. Three days after modeling, the DNT cell suspensions (Perio+DNT Low and Perio+DNT High) were locally injected into the gingiva of the teeth with periodontitis (the maxillary right second molar), denoted as DNT Low and DNT High. In addition, a normal mouse control group (Control) injected with 5 μl of normal saline and a periodontitis mouse control group (Periodontitis) injected with 5 μl of normal saline were set up.

[0064] On the 4th day after injection, the maxillary bone samples were harvested and subjected to Micro-CT scanning. The software measured the distance from the Cementum Enamel Junction (CEJ) to the Alveolar Bone Crest (ABC) to evaluate the resorption of alveolar bone. The results are shown in Figure 5 and Figure 6 ; After obtaining the tissues, they were fixed with 4% paraformaldehyde, decalcified with 10% EDTA, embedded in paraffin, sectioned and stained with HE to observe inflammation and bone destruction; Gingival tissues were obtained for transcriptome sequencing and RT-PCR verification of the mRNA expression levels of IL-1β, TNFα, CCL2 and IL-17A, with GAPDH as the internal reference gene. The results are shown in Figure 7 , and the primer sequences are shown in Table 1.

[0065] Table 1 Amplification primer sequences of different genes

[0066]

[0067] It can be seen from Figures 5 - 6 that local injection of TCRαβ+DNT cells at different doses (1000 and 5000) into mice all showed a significant reduction in periodontal bone resorption and destruction (309.1±26.68 μm in periodontitis mice, 249.1±20.44 μm in the DNT Low group, and 278.2±19.86 μm in the DNT High group), and the treatment effect was better with 1000 cells.

[0068] It can be seen from Figure 7It can be seen that the mRNA expression levels of inflammatory factors IL-1β, TNFα, CCL2, and IL-17A in the periodontal tissue were significantly decreased. Among them, for IL-1β in periodontitis mice: 1.04 ± 0.32, and in DNT Low-treated mice: 0.3 ± 0.13; for TNFα in periodontitis mice: 1.01 ± 0.13, and in DNT Low-treated mice: 0.48 ± 0.08; for CCL2 in periodontitis mice: 1.06 ± 0.41, and in DNT Low-treated mice: 0.35 ± 0.11; for IL-17A in periodontitis mice: 1.03 ± 0.47, and in DNT Low-treated mice: 0.51 ± 0.18.

[0069] Example 5

[0070] Perforin knockout mice (denoted as prf1 - / - ) were purchased from Jackson Laboratory (Jackson Lab) in the United States (denoted as prf1 - / - ).

[0071] Using the method of Example 4, wild-type mice (C57BL / 6 mice) were treated by ligating the maxillary second molar with 4-0 silk thread to construct periodontitis model mice (denoted as Perio).

[0072] Using the method of Example 4, TCRαβ - / - DNT cells of perforin knockout mice (prf1 + ) and wild-type mice were isolated to obtain two DNT cell suspensions with a concentration of 1000 cells / 5 μl, which were denoted as prf1 - / - DNT and DNT, respectively.

[0073] Using the method of Example 4, mice with periodontitis were treated by injecting at a dose of 1000 DNT cells / mouse; in addition, a control group of periodontitis mice (Perio) injected with 5 μl of normal saline was set up. Micro-CT scanning was performed, and software was used to measure the distance from the cementoenamel junction to the alveolar crest top to evaluate the alveolar bone destruction and resorption; gingival tissue was obtained, fixed, embedded, and immunofluorescence staining was performed to detect the neutrophil marker Ly6G, the cytokine IL-1b, and the extracellular trap marker citrullinated histone H3 (Cit-H3). The results are shown in Figures 8 - 14 .

[0074] Perforin is a functional molecule of TCRαβ+ DNT cells. The protective effect of TCRαβ+ DNT cells against periodontitis is mainly achieved through the perforin molecule. By comparing the TCRαβ+ DNT cells of wild-type and perforin-knockout mice, it was found that the protective effect of TCRαβ+ DNT cells derived from perforin-knockout mice against periodontitis was significantly reduced ( Figures 8 - 9 ). The situation of periodontal bone resorption and destruction is as follows: for periodontitis mice, it was 303.8 ± 21.7 μm; for the DNT treatment group, it was 246 ± 19.4 μm; for the prf1 - / - DNT treatment group, it was 290.2 ± 15.19 μm.

[0075] Neutrophils are important pathogenic immune cells in periodontitis. Through immunofluorescence staining of periodontal tissues, it was found that in the periodontal tissues treated with DNT cells, the absolute number (average value counted per field of view) of neutrophils was significantly lower than that of untreated periodontal tissues. Among them, for periodontitis mice, it was 19.4 ± 2.97; for the DNT treatment group, it was 11.6 ± 2.7; for the prf1 - / - DNT treatment group, it was 16 ± 2.24 ( Figures 10 - 11 ). And the proportion of neutrophils secreting IL-1β decreased significantly. Among them, for periodontitis mice, it was 66 ± 5.87%; for the DNT treatment group, it was 50 ± 6.67%; for the prf1 - / - DNT treatment group, it was 60.2 ± 6.3% ( Figure 10 ), and the proportion of neutrophils secreting neutrophil extracellular traps also decreased significantly ( Figure 12 ). Among them, for periodontitis mice, it was 41.8 ± 5.76%; for the DNT treatment group, it was 26 ± 3.54%; for the prf1 Figures 13 - 14 DNT treatment group, it was 32.6 ± 4.28%. - / - DNT treatment group, it was 32.6 ± 4.28%.

[0076] In summary, the TCRαβ+ DNT cells prepared by the present invention do not need to be amplified and cultured in vitro and can be directly used for local injection of autologous tissues, achieving a high proportion of TCRαβ+ DNT cell levels locally, thereby achieving the effects of preventing and treating autoimmune diseases and inflammatory diseases.

[0077] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing double-negative T cells, characterized in that: The following steps are involved: Separating mononuclear cells from peripheral blood or lymphoid tissue to obtain mononuclear cell populations; CD4 T cells, CD8 T cells, NK cells, granulocytes, monocyte macrophages, gamma-delta T cells and B cells in the mononuclear cell population are removed to obtain double negative T cells.

2. The preparation method according to claim 1, characterized in that: The reagents used for the separation include lymphocyte separation fluid.

3. The preparation method according to claim 1, characterized in that: The removal method includes an immunomagnetic bead method.

4. The preparation method according to claim 3, characterized in that: The antibodies used in the immunomagnetic bead method include: CD4 antibody, CD8α antibody, CD8β antibody, NK antibody, CD11b antibody, CD14 antibody, TCRγδ antibody and CD19 antibody; the NK antibody is NK1.1 or CD56.

5. The preparation method according to claim 4, characterized in that: The antibody is a PE-labeled antibody, a FITC-labeled antibody or a Biotin-labeled antibody.

6. The preparation method according to claim 1, characterized in that: The lymphatic tissue includes the spleen or lymph nodes.

7. Use of the double negative T cells prepared by the preparation method according to any one of claims 1 to 6 in the preparation of products for preventing and treating autoimmune diseases and / or inflammatory diseases.

8. The use according to claim 7, characterized in that: The inflammatory disease includes one or more of periodontitis, arthritis, spinal cord injury and spinal cord inflammation.

9. A preparation for preventing and treating periodontitis, characterized in that: The effective ingredients of the preparation include double-negative T cells; the double-negative T cells are double-negative T cells prepared by the preparation method according to any one of claims 1 to 6.

10. The preparation according to claim 9, characterized in that The dosage form of the preparation includes an injection; the solvent of the injection includes physiological saline.

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