CN110731968A

CN110731968A - ICOS+CXCR3+Application of regulatory T cells in preparation of severe pneumonia prevention medicine

Info

Publication number: CN110731968A
Application number: CN201910851047.1A
Authority: CN
Inventors: 孙凌云; 李文超; 陈纬纬
Original assignee: Nanjing Drum Tower Hospital
Current assignee: Nanjing Drum Tower Hospital
Priority date: 2019-09-10
Filing date: 2019-09-10
Publication date: 2020-01-31
Also published as: WO2021047144A1

Abstract

The invention discloses ICOS⁺CXCR3⁺Application of regulatory T cells in preparation of severe pneumonia prevention medicines. The pneumonia preventing medicine comprises ICOS⁺CXCR3⁺Regulatory T cells and a pharmaceutically acceptable carrier. The invention has the advantages of good prevention and protection effect, lasting time and the like.

Description

ICOS+CXCR3+Application of regulatory T cells in preparation of severe pneumonia prevention medicine

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to ICOSs⁺CXCR3⁺Application of regulatory T cells in preparation of severe pneumonia prevention medicines.

Background

Severe pneumonia is progressive pulmonary inflammation, which can be rapidly evolved from local infection to systemic infection, severe sepsis, septic shock, Multiple Organ Dysfunction Syndrome (MODS), among all hospitalized pneumonia patients, 10% -20% of patients with severe pneumonia need to stay in an intensive care unit for treatment, because the disease has a high mortality rate, the treatment of severe pneumonia is a hot spot of research in the academia, the research shows that the death of most pneumonia patients occurs after bacterial clearance, and the death of pneumonia patients is not enough to reduce the death rate of pneumonia patients by anti-infection treatment alone, the cause of the death is series immune response caused by pathogenic bacteria invasion, a cascade effect is generated, the local and systemic persistent inflammatory response causes Multiple Organ Dysfunction Syndrome (MODS), the pathogenesis of severe pneumonia is not detailed at present, the pathological changes mainly include activation of cells, increase of inflammatory mediators, enhancement of leukocytes and cells, the proliferation of cells in endothelial cells, and the activation of cells, the systemic inflammatory response is an important therapeutic factor for the systemic inflammatory response, the systemic inflammatory response is an anti-inflammatory response of the lung, and the inflammatory response of the lung is an important therapeutic strategy for the increase of the systemic inflammatory response of the lung.

The conventional treatment methods for severe pneumonia include:

(1) unlike antibiotic escalation therapy for mild pneumonia, antibiotic therapy for severe pneumonia requires early, combined, and escalating principles, the earlier an effective antibiotic therapy should be better, the later use of antibiotics should be delayed, the prognosis of patients is unfavorable, and mortality is increased, studies have shown that severe pneumonia patients have a significantly reduced mortality within 4 hours of antibiotic administration, as compared to 4 hours later antibiotic administration, Diaz-Martin et al demonstrate that severe patients have a significantly reduced mortality as compared to single antibiotic administration, and that Thoracic American Society of sciences (American Thoracic Society, ATS) guidelines recommend intravenous use of β -lactams, macrolides, or quinolones, if patients are allergic to penicillin, the selection of quinolones and aztreonam, if considered as a pseudomonas aeruginosa infection, the use of pseudomonas aeruginosa-resistant β -lactam drugs plus or levofloxacin antibiotics, or the increased risk of cardiovascular mortality as indicated by MRS, and further consider the use of antibiotics.

(2) Glucocorticoid inhibits certain links of the inflammatory process by altering gene transcription, such as reducing transcription of proinflammatory factors, chemokines, adhesion molecules, intracellular inflammatory receptors, inhibiting migration of polymorphonuclear cells within the blood vessels, and by reducing the expression of various proinflammatory enzymes, resulting in a down-regulation of inflammatory responses, and by reducing the production of inflammatory substances by reducing the expression of various proinflammatory enzymes, it has been found that glucocorticoid receptor complexes down-regulate the gene transcription of proinflammatory cytokines such as interleukin (interleukin, IL) -1, gamma-interferon, IL-2, IL-8, etc., by interacting directly with glucocorticoid response elements on DNA or by inhibiting the activity of two important transcription factor activator proteins-1 and nuclear factor-kb, while promoting the production of anti-inflammatory factor IL-10, suggesting that the protective effects of the hormone may be balanced with its proinflammatory mediators/mediators, anti-inflammatory mediators, and the effects of inflammatory mediators are shown to be a large-scale review, and even though the acute adverse effects of glucocorticoid therapy may be seen in acute sepsis, the acute inflammatory response, the acute risk of sepsis is a review of acute sepsis.

(3) Vasoactive drug therapy: microcirculation disturbance is the pathological basis of severe pneumonia and multiple organ dysfunction, and correct application of vasoactive drugs can improve and activate microcirculation, change low perfusion of important organs, and play an important role in preventing severe pneumonia and complications thereof from developing and reversing the state of an illness. The clinical application of more vasoactive drugs mainly comprises dopamine, and according to the conditions of illness and complications, the vasoactive drugs such as dobutamine, phentolamine and Dong scopolamine are used singly or in a combined mode.

Disclosure of Invention

The invention provides ICOSs⁺CXCR3⁺The application of regulatory T cells in preparing a medicament for preventing severe pneumonia overcomes the defects of the prior art.

To achieve the above object, the present invention provides ICOSs⁺CXCR3⁺The application of regulatory T cells in preparing a medicament for preventing severe pneumonia.

step, the invention provides ICOSs⁺CXCR3⁺The use of regulatory T cells may also be characterized by: wherein the pneumonia preventing drug comprises ICOS⁺CXCR3⁺Regulatory T cells and a pharmaceutically acceptable carrier.

step, the invention provides ICOSs⁺CXCR3⁺The use of regulatory T cells may also be characterized by: wherein, the pharmaceutical preparation for preventing pneumonia is injection.

step, the invention provides ICOSs⁺CXCR3⁺The use of regulatory T cells may also be characterized by: wherein the ICOS⁺CXCR3⁺The separation method of regulatory T cells comprises the steps of , separating mouse spleen mononuclear cells to obtain mouse spleen mononuclear cell suspension, secondly, sorting CD4T cells in the mouse spleen mononuclear cell suspension through magnetic beads to obtain CD4T cell suspension, and thirdly, sorting ICOS in the CD4T cell suspension in a flow mode⁺CXCR3⁺Regulatory T cells.

step, the invention provides ICOSs⁺CXCR3⁺The application of regulatory T cells can also be characterized in that the specific process of the step is to kill a mouse by removing cervical vertebra, soak the mouse in 75% alcohol for sterilization, then take out and fix the mouse on a dissecting plate of the mouse, cut the mouse perineum and scratch the head, peel off the skin for fixation, cut the mouse abdominal envelope, take out the spleen, add precooled PBS buffer solution, grind tissue fragments by wool glass, filter the tissue fragments by a nylon membrane of 200 meshes to prepare single cell suspension, centrifuge for 5 minutes at 4 ℃ and 1650rpm, discard the supernatant, flick the spleen cells, add 2mL of erythrocyte lysate, blow and mix the cells evenly, stand the cells for 2 minutes at room temperature, add 10mL of PBS buffer solution to stop the reaction, centrifuge for 5 minutes at 1650rpm and 4 ℃, discard the supernatant, resuspend and wash the cells by the PBS buffer solution again, centrifuge, discard the supernatant, resuspend the cells by 4mL of complete cell culture solution, add 10 mul cell suspension into 90 mul trypan blue, count and adjust the cell concentration to a working concentration of 1 × 10⁶/mL。

step, the invention provides ICOSs⁺CXCR3⁺The use of regulatory T cells may also be characterized by: wherein, the specific process of the step two is as follows: taking spleen cell suspension, centrifuging the cells at 300g and 4 ℃, and removing supernatant; according to each 10⁷Adding 90 mul MACS buffer solution into each cell, and adding the buffer solution and then resuspending the cells; according to each 10⁷Adding 10 mu lCD4(L3T4) microbeads to each cell, and adding the microbeads to the cell suspension; gently and uniformly blowing and beating, and incubating for 15 minutes at 4 ℃; according to each 10⁷Adding 2ml of MACS buffer solution into each cell, adding buffer solution to wash the cells, taking 300g, and separating at 4 DEG CHeart cells, and supernatant was removed; add 500. mu.l buffer to resuspend the cells and transfer to MACS buffer equilibrated and mounted on a magnet rack LS column; the LS column was washed 3 times with 5ml of MACS buffer each time; taking the LS column off the magnet frame, placing the LS column on a 15ml centrifuge tube, adding 5ml of MACS buffer solution into the LS column, forcibly pushing the liquid out of the LS column by a piston, and collecting the effluent, namely the purified CD4T cell suspension; centrifuging the cells at 4 deg.C at 300g, removing supernatant, and resuspending the cells in PBS to a concentration of 2X 10⁷pieces/mL, put on ice for use.

step, the invention provides ICOSs⁺CXCR3⁺The use of regulatory T cells may also be characterized by: the specific process of the third step is as follows: staining and marking the separated CD4T cells with anti-CD 25-APC, anti-CD 4-FITC, anti-ICOS-PerCP-Cy5.5 and anti-CXCR 3-PE monoclonal antibodies, and collecting ICOS by BD-ARIA sorting⁺CXCR3⁺Regulatory T cells.

step, the invention provides ICOSs⁺CXCR3⁺The use of regulatory T cells may also be characterized by: wherein the ICOS⁺CXCR3⁺The in vitro expansion culture process of the regulatory T cells comprises the following steps: will separate the ICOS⁺CXCR3⁺Regulatory T cells were seeded in 96-well U plates at 5X 10 per well⁴Individual cells, 5% CO at 37 ℃₂Culturing the cells with 200 ul of 10% calf serum RPMI1640 medium under saturated humidity, wherein the culture system also contains 2mmol/L glutamine, 25mmol/LHEPES, 50U/mL penicillin, 50 ug/mL streptomycin, 50 ul mol/L2-mercaptoethanol, 100nmol/L rapamycin, 400U/mL interleukin-2 and mouse T cell amplification magnetic beads, the number of the magnetic beads is 4 times of the number of the cells at , the ratio of the magnetic beads to the cells is 1:1, observing the growth condition of the cells under a microscope, changing the liquid for 1 time and 1:3 passages for about 3 days, supplementing the medium with fresh RPMI1640 containing the components to 200 ul, and culturing the collected cells after 2-3 weeks.

The invention has the beneficial effects that ICOSs are provided by the invention⁺CXCR3⁺Application of regulatory T cells in preparation of severe pneumonia prevention medicine, ICOS⁺CXCR3⁺The basic research of the ICOS + CXCR3+ Treg cells is solid at present, the technical thrust degree is high, the technology is controllable, the safety is good, the acceptance degree of treatment cost is good, and the benefit is . therefore, the application of the ICOS + CXCR3+ Treg cells in preparing the medicine for treating the severe pneumonia has huge social and economic benefits.

Drawings

FIG. 1 is an ICOS⁺CXCR3⁺Proliferation profiles of regulatory T cells co-cultured with PBMCs and PBMC alone;

FIG. 2 is an ICOS⁺CXCR3⁺A map of the expression of each molecule on the surface of regulatory T cells;

FIG. 3 is a graph comparing the pulmonary bronchoalveolar lavage fluid and the H.influenzae load in the lung after infection with H.influenzae at different times in various groups of mice;

FIG. 4 is a graph comparing the content of inflammatory cytokines in the lung (IL-6, TNF- α, KC, MIP-2 and MCP-1) in lung bronchoalveolar lavage fluid after infection with Haemophilus influenzae at different times in groups of mice;

FIG. 5 is a photograph of a tissue section of lung after infection of various groups of mice with Haemophilus influenzae at various times.

Detailed Description

Regulatory T cells refer to CD4⁺CD25⁺T cells are produced in the thymus and exported to the periphery. ICOS and CXCR3 are molecules expressed on regulatory T cells.

The invention is further illustrated in with reference to specific examples.

Example 1

ICOS⁺CXCR3⁺In vitro isolation and expansion culture of regulatory T cells

ICOS⁺CXCR3⁺Isolation of regulatory T cells

Step , separating mouse spleen mononuclear cells to obtain mouse spleen mononuclear cell suspension, wherein the specific process comprises the following steps:

1) material and reagent preparation: the kit comprises a mouse dissecting instrument, 75% alcohol, sterile PBS buffer solution, complete culture solution of mouse spleen and lung cells (10% calf serum RPMI1640 culture medium), erythrocyte lysate and a 200-mesh nylon membrane.

2) And taking off the cervical vertebra, killing the mouse, soaking the mouse in 75% alcohol, disinfecting for 10 minutes, taking out and fixing the mouse on a mouse dissection plate.

3) Cutting the perineum of the mouse with an ophthalmological scissors and scratching the perineum to the head, peeling off the outer skin for fixation, cutting the abdominal envelope, taking out the spleen by matching the ophthalmological scissors and forceps, adding a proper amount of precooled PBS buffer solution, gently grinding tissue fragments with a wool glass, and filtering with a 200-mesh nylon membrane to prepare single cell suspension.

4) The mixture was centrifuged at 1650rpm at 4 ℃ for 5 minutes, and the supernatant was discarded.

5) Gently bouncing splenocytes, adding 2ml of erythrocyte lysate, blowing and mixing evenly, and standing for 2 minutes at room temperature.

6) After the reaction was terminated by adding 10ml of PBS buffer, the mixture was centrifuged at 1650rpm and 4 ℃ for 5 minutes, and the supernatant was discarded.

7) The cells were resuspended in PBS buffer and washed, centrifuged, and the supernatant discarded.

8) 4mL of the cell complete culture solution, resuspending the cells, adding 10. mu.l of the cell suspension to 90. mu.l of trypan blue, counting and adjusting the cell concentration to a working concentration of 1X 10⁶and/mL, obtaining the mouse spleen mononuclear cell suspension.

And step two, sorting CD4T cells by magnetic beads in mouse spleen mononuclear cell suspension to obtain CD4T cell suspension. The specific process is as follows:

1) the spleen cell suspension was collected, centrifuged at 300g and 4 ℃ to remove the supernatant.

2) According to each 10⁷The cells were resuspended after addition of the buffer at a rate of 90. mu.l MACS buffer.

3) According to each 10⁷The cells were added to a cell suspension at a rate of 10. mu.l of CD4(L3T4) beads.

4) The mixture was gently and evenly blown and incubated at 4 ℃ for 15 minutes.

5) Every 10 according to⁷The cells were washed by adding 2ml of MACS buffer to each cell, and then the cells were centrifuged at 4 ℃ at 300g to remove the supernatant.

6) The cells were resuspended by adding 500. mu.l buffer and transferred to a LS column equilibrated with MACS buffer and mounted on a magnet holder.

7) The LS column was washed 3 times with 5ml of MACS buffer.

8) The LS column was removed from the magnet holder and placed in a 15ml centrifuge tube, 5ml of MACS buffer was added to the LS column, the liquid was pushed out of the LS column rapidly with force by a plunger, and the effluent was collected as purified CD4T cell suspension.

9) Centrifuging the cells at 4 deg.C at 300g, removing supernatant, and resuspending the cells with PBS to a concentration of 2 × 10⁷pieces/mL, put on ice for use.

Step three, in CD4T cell suspension, flow sorting ICOS⁺CXCR3⁺Regulatory T cells. The specific process is as follows: staining and marking the separated CD4T cells with anti-CD 25-APC, anti-CD 4-FITC, anti-ICOS-PerCP-Cy5.5 and anti-CXCR 3-PE monoclonal antibodies, and collecting ICOS by BD-ARIA sorting⁺CXCR3⁺Regulatory T cells.

Two, ICOS⁺CXCR3⁺In vitro expansion culture of regulatory T cells

Will separate the ICOS⁺CXCR3⁺Regulatory T cells were seeded in 96-well U plates at approximately 5X 10 per well⁴Individual cells, 5% CO at 37 ℃₂2mmol/L glutamine, 25mmol/L HEPES, 50U/mL penicillin, 50. mu.g/mL streptomycin, 50. mu. mol/L2-mercaptoethanol, 100nmol/L rapamycin, 400U/mL interleukin-2 (IL-2), and mouse T cell expansion magnetic beads, wherein the number of the magnetic beads at is 4 times of the number of the cells, and the ratio to the cells is 1:1, and a cell culture solution containing the IL-2 and the T cell expansion magnetic beads except the above is also used in the following cell culture experimentNext, passage 1:3, the medium was supplemented to 200. mu.l with fresh RPMI1640 containing the above ingredients. And collecting cells after culturing for 2-3 weeks.

Three, ICOS⁺CXCR3⁺Detection of regulatory T cells

1、ICOS⁺CXCR3⁺Regulatory T cell activity and purity assays

ICOS⁺CXCR3⁺The regulatory T cell suspension was stained with 10. mu.l of Trypan blue, 200 cells were counted under the microscope, and the viable cell rate was calculated. And taking 100 mu l of cell suspension, adding CD4, CXCR3, ICOS and CD25 antibodies, and detecting the purity of the cells by using a flow cytometer. The living cell rate and the cell purity are both more than 95 percent.

2、ICOS⁺CXCR3⁺Regulatory T cell suppression function

Collection of fresh and expanded ICOS⁺CXCR3⁺Regulatory T cells (Tregs), counted, 1X 10⁵More than one cell was irradiated (3000rads) with 2X 10 cells⁵Co-culture of xenogenic (human) PBMCs in 96-well U-plates, and 2X 10 irradiated (3000rads)⁵The heterogeneous (human) PBMCs are separately cultured in another 96-hole U-shaped plate at 37 ℃ and 5% CO₂After co-culturing in incubator for 5 days, the degree of cell proliferation was measured using WST-1 cell proliferation kit, and the results are shown in FIG. 1, ICOS⁺CXCR3⁺Regulatory T cells can significantly inhibit peripheral blood mononuclear cell proliferation.

Example 2

ICOS⁺CXCR3⁺Method of administering regulatory T cells

, final authentication before use

1. Sterility test

If no abnormality occurs during the culturing process, the method is conventionally performed in ICOS⁺CXCR3⁺Two days before the infusion of regulatory T cells into mice, and finally liquid changes, the supernatant in each culture dish is subjected to sterility test to culture bacteria and mold, and the cell supernatant cannot grow bacteria and mold.

2. Appearance and under-mirror observation

On the day of infusion, the supernatant in the dish was color-observed for cell morphology, cell density, presence or absence of bacteria or mold growth under an inverted microscope as sterility tests reported no bacteria, mold growth. If suspicious, sampling again to culture bacteria and mold for sterility test.

3. Proportion of viable cells

The collected cell suspension was sampled on the day of infusion to check the proportion of viable cells to be more than 95%.

4. Karyotype of chromosome

ICOS⁺CXCR3⁺Samples were taken 2 days prior to regulatory T cell infusion for karyotyping and the results were normal.

5. Surface sign

Surface labeling assay of sample rows 2 days prior to infusion: as a result, as shown in FIG. 2, CD4, CD25, ICOS, CXCR3 and CTLA-4 were expressed positively.

Two, ICOS⁺CXCR3⁺Method of administering regulatory T cells

1. Preparation of the apparatus, materials and reagents used

Clean bench, centrifuge, disposable syringe.

2. Pre-infusion procedures

When the cells reached 1X 10⁶Mice, 20g, were ready for infusion. I.e. by 1 x 10⁶Mice were infused with 20g of mouse body weight. Before administration, cells were taken from the clean bench and cell concentration, proportion of viable cells, cell surface markers, and cell karyotype were determined by counting.

3. Infusion operation

1) The mixture was centrifuged (170 g at room temperature for 5 minutes), and the supernatant was discarded.

2) The cells were transferred to a centrifuge tube, PBS was added, the mixture was mixed well and centrifuged, the supernatant was discarded, and the procedure was repeated times.

3) After washing, 200. mu.l of PBS was added to resuspend the cells, and 20. mu.l of the cell suspension was taken to count the number of cells.

4) Mice were infused with cells via tail vein.

5) And observing the reaction of the mice in the infusion process, and timely carrying out symptomatic treatment.

Example 3

ICOS⁺CXCR3⁺Application of regulatory T cells in preparation of medicine for preventing severe bacterial pneumonia animal model

1. Preparation of haemophilus influenzae infected bacterial liquid

1) Taking out the haemophilus influenzae NT127 frozen at-80 ℃, dipping a little by using a sterilized wood stick, streaking and inoculating the dipped small into an sBHI culture plate, and culturing the small in a carbon dioxide incubator at 37 ℃ overnight.

2) And a plurality of single colonies were picked and inoculated into 10ml of sBHI liquid medium, and cultured at 37 ℃ in a shaker at 130rpm until OD600 is 0.4-0.5.

3) The cells were centrifuged at 4000rpm for 15 minutes at room temperature to collect the bacteria.

4) 12ml of PBS buffer, the bacteria were resuspended, centrifuged at 4000rpm at room temperature for 15 minutes, and the bacteria were collected. The PBS buffer was resuspended and OD600 was adjusted to 1.0, at which time the bacterial concentration was approximately 3X 109 CFU/ml.

5) Diluting with 10-fold gradient, taking 10-6 dilution, sucking 10 μ l of bacterial liquid to sBHI culture plate, spotting for three times, and after the bacterial liquid is completely absorbed by the plate, CO at 37 deg.C₂And (5) an incubator is inverted and cultured overnight, colonies are counted the next day, and the bacterial concentration is accurately calculated.

2. Mouse haemophilus influenzae infection model

Injecting 100 μ l/20g anesthetic into abdominal cavity of mouse, placing the mouse on the palm of hand after anesthesia, and slowly dripping 30 μ l (about 1 × 10) of bacteria via right nasal wing by pipette with respiratory rhythm of mouse⁸CFU). When the mouse is not choked, the mouse is placed in the squirrel cage in a side lying mode. Upon confirmation of mouse awakening, the mouse was left. Establishing mouse hemophilus influenzae lung infection.

3、ICOS⁺CXCR3⁺Prevention of mouse infected Haemophilus influenzae by regulatory T cells

Taking eight groups of mice, wherein four groups of mice are according to ICOS⁺CXCR3⁺Method of administering regulatory T cells "infusion of ICOS⁺CXCR3⁺Regulatory T cells; the other four mice were blank control group and were not infused with ICOS⁺CXCR3⁺Regulatory T cells.

In infusion of ICOS⁺CXCR3⁺Eight groups of mice were infected with Haemophilus influenzae on th, seventh, and fourteenth days after the regulatory T cells, respectively, according to the method of "mouse Haemophilus influenzae infection model".

FIG. 3 is a graph showing the comparison of the Haemophilus influenzae load in lung and pulmonary bronchoalveolar lavage fluid after infection with Haemophilus influenzae at different times in each group of mice. As shown in FIG. 3, ICOS was infused⁺CXCR3⁺Groups of mice with regulatory T cells had lower lung bronchoalveolar lavage fluid and lower H.influenzae loads in the lungs than uninfected ICOS⁺CXCR3⁺Groups of mice with regulatory T cells (blank control).

FIG. 4 is a graph showing a comparison of the content of inflammatory cytokines (IL-6, TNF- α, KC, MIP-2 and MCP-1) in lung bronchoalveolar lavage fluid after infection with Haemophilus influenzae at different times in mice in each group, and ICOS infusion is performed as shown in FIG. 4⁺CXCR3⁺The concentration of inflammatory cytokines in lung bronchoalveolar lavage fluid of each group of mice with regulatory T cells was lower than that of uninformed ICOS⁺CXCR3⁺Groups of mice with regulatory T cells (blank control).

FIG. 5 is a photograph of a lung tissue section obtained after infection of various groups of mice with Haemophilus influenzae at various times. As shown in FIG. 5, ICOS was infused⁺CXCR3⁺Mice with regulatory T cells with significantly lower lung inflammatory cell infiltration than uninjected ICOS⁺CXCR3⁺Mice with regulatory T cells.

Thus, with ICOS⁺CXCR3⁺The medicine preparation with regulatory T cells as active component can prevent severe pneumonia, and has good prevention and protection effect and long-lasting time.

Claims

1, ICOS⁺CXCR3⁺Use of regulatory T cells characterized by: can be used for preparing medicine for preventing severe pneumonia.

2. Use according to claim 1, characterized in that:

wherein the medicament for preventing pneumonia comprises ICOS⁺CXCR3⁺Regulatory T cells and a pharmaceutically acceptable carrier.

3. Use according to claim 1, characterized in that:

wherein the pharmaceutical preparation for preventing pneumonia is an injection.

4. Use according to claim 1, characterized in that:

wherein the ICOS⁺CXCR3⁺The method for separating the regulatory T cells comprises the following steps:

step , separating mouse spleen mononuclear cells to obtain a mouse spleen mononuclear cell suspension;

step two, sorting CD4T cells by magnetic beads in the mouse spleen mononuclear cell suspension to obtain a CD4T cell suspension;

step three, in the CD4T cell suspension, flow sorting ICOS⁺CXCR3⁺Regulatory T cells.

5. Use according to claim 4, characterized in that:

the specific process of the step is as follows:

removing cervical vertebrae, killing the mouse, soaking in 75% alcohol for disinfection, and taking out and fixing on a mouse dissection plate;

cutting a mouse perineum part and scratching the mouse head part, peeling off the outer skin for fixation, cutting a mouse abdominal envelope part, taking out the spleen, adding precooled PBS buffer solution, grinding tissue fragments by using a wool slide, and filtering by using a 200-mesh nylon membrane to prepare single cell suspension;

centrifuging at 4 deg.C and 1650rpm for 5 min, and removing supernatant;

gently bouncing splenocytes, adding 2ml of erythrocyte lysate, blowing and uniformly mixing, and standing for 2 minutes at room temperature;

adding 10ml PBS buffer solution to stop reaction, centrifuging for 5 minutes at 1650rpm and 4 ℃, and removing supernatant;

resuspending and washing the cells again by using the PBS buffer solution, centrifuging, and discarding the supernatant;

resuspend cells in 4mL of complete cell culture, add 10. mu.l of cell suspension to 90. mu.l of Trypan blue, count and adjust the cell concentration to 1X 10 working concentration⁶/mL。

6. Use according to claim 4, characterized in that:

the specific process of the second step is as follows:

taking spleen cell suspension, centrifuging the cells at 300g and 4 ℃, and removing supernatant;

according to each 10⁷Adding 90 mul MACS buffer solution into each cell, and adding the buffer solution and then resuspending the cells;

according to each 10⁷Adding 10 mu lCD4(L3T4) microbeads to each cell, and adding the microbeads to the cell suspension;

gently and uniformly blowing and beating, and incubating for 15 minutes at 4 ℃;

according to each 10⁷Adding 2ml of MACS buffer solution into each cell, adding the buffer solution to wash the cells, then taking 300g of the cells, centrifuging the cells at 4 ℃, and removing the supernatant;

add 500. mu.l buffer to resuspend the cells and transfer to MACS buffer equilibrated and mounted on a magnet rack LS column;

the LS column was washed 3 times with 5ml of MACS buffer each time;

taking the LS column off the magnet frame, placing the LS column on a 15ml centrifuge tube, adding 5ml of MACS buffer solution into the LS column, forcibly pushing the liquid out of the LS column by a piston, and collecting the effluent, namely the purified CD4T cell suspension;

centrifuging the cells at 4 deg.C at 300g, removing supernatant, and resuspending the cells in PBS to a concentration of 2X 10⁷pieces/mL, put on ice for use.

7. Use according to claim 4, characterized in that:

the third step comprises the following specific processes: the separated CD4T cells were treated with anti-CD 25-APC, anti-CD 4-FITC, anti-ICOS-PerCP-Cy5.5 and anti-CXCR 3-PE monoclonal antibody for staining and labeling, and collecting ICOS by BD-ARIA sorting⁺CXCR3⁺Regulatory T cells.

8. Use according to claim 4, characterized in that:

wherein the ICOS⁺CXCR3⁺The in vitro expansion culture process of the regulatory T cells comprises the following steps:

separating the ICOS⁺CXCR3⁺Regulatory T cells were seeded in 96-well U plates at 5X 10 per well⁴Individual cells, 5% CO at 37 ℃₂Culturing the cells in 200. mu.l of 10% calf serum RPMI1640 medium under saturated humidity conditions;

the culture system also contains 2mmol/L glutamine, 25mmol/L HEPES, 50U/mL penicillin, 50 mu g/mL streptomycin, 50 mu mol/L2-mercaptoethanol, 100nmol/L rapamycin, 400U/mL interleukin-2 and mouse T cell amplification magnetic beads;

the number of the magnetic beads at week was 4 times the number of the cells, and then the ratio of the number of the magnetic beads to the number of the cells was 1: 1;

observing cell growth under microscope, changing solution for about 3 days for 1 time, passaging at 1:3, and supplementing culture medium with fresh RPMI1640 containing above components to 200 μ l;

and collecting cells after culturing for 2-3 weeks.