CN115404241A

CN115404241A - Preparation method and application of extracellular vesicles derived from genetically engineered regulatory T cells

Info

Publication number: CN115404241A
Application number: CN202210965005.2A
Authority: CN
Inventors: 陈江华; 林锦雯
Original assignee: First Affiliated Hospital of Zhejiang University School of Medicine
Current assignee: First Affiliated Hospital of Zhejiang University School of Medicine
Priority date: 2022-08-12
Filing date: 2022-08-12
Publication date: 2022-11-29

Abstract

A preparation method and application of an extracellular vesicle derived from genetically engineered regulatory T cells belong to the technical field of biology. The invention provides a preparation method of a genetically engineered regulatory T cell and a method for using the T cellA preparation method and application of extracellular vesicles. The preparation method of the genetically engineered regulatory T cell provided by the invention can overcome the practical problem of low source yield of the existing regulatory T cell, and can obtain the CD4 with simple source and high yield ⁺ CD25 ^‑ T cells are transfected by lentivirus particles, so that a stable cell line for expressing the FOXP3 gene in high purity is obtained; the method for preparing and separating the extracellular vesicles can separate the vesicle concentrated solution with less protein pollution and high activity, is based on a method combining a size exclusion method and rotary ultrafiltration, has high separation efficiency and high speed, ensures the separation purity by combining double methods, greatly reduces pollution and reduces the adverse reaction of a user.

Description

Preparation method and application of extracellular vesicles derived from genetically engineered regulatory T cells

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of an extracellular vesicle derived from a genetically engineered regulatory T cell.

Background

Regulatory T cells are a subset of T cells with significant immunosuppressive effects, expressing FOXP3, CD25, CD4 as the phenotypic characteristics of the cells. It can inhibit the immune response of other cells, is the main controller of self tolerance, and has important significance in regulating the immune homeostasis of the body and preventing autoimmune diseases. In view of its immunosuppressive and tolerogenic functions, regulatory T cells play an important role in the field of alleviating organ transplant rejection and the treatment of autoimmune diseases. Meanwhile, regulatory T cells have obvious anti-inflammatory action, can secrete anti-inflammatory cytokines such as IL-4, IL-10, TGF-beta and the like to inhibit autoinflammation reaction, and prevent pathological immune response causing tissue damage.

Depending on the source of regulatory T cells, there may be a distinction between natural regulatory T cells and adaptive regulatory T cells. Natural regulatory T cells are predominantly CD4 ⁺ CD25 ⁺ Regulatory T cells, differentiated in the thymus from bone marrow-derived progenitor cells, exert an immunomodulatory effect in peripheral blood, lymphoid organs, inflammatory sites and tumor tissues; the adaptive regulatory T cells are formed by the development of peripheral naive T cells under the induction of small-dose antigens or immunosuppressive cytokines, comprise cells such as Tr1, th3 and the like, and mainly secrete IL-10 and TGF-beta to play a role in immune negative regulation. CD4 ⁺ CD25 ⁺ Regulatory T cells have proven to be of value in the treatment of autoimmune diseases and in the induction of transplantation tolerance, but their clinical use is greatly limited because they account for only 5% -10% of the total number of CD4+ T lymphocytes in the peripheral blood. For this reason, many researchers have tried in vitro culture, amplification and have made some progress, such as stimulation of CD4 with anti-CD 28 monoclonal antibody, anti-CD 3 monoclonal antibody, large doses of IL-2, IL-10 and IFN ⁺ CD25 ⁺ Tregs, which are allowed to expand in vitro. For example, patent publication No. CN102459577A discloses a method for in vitro expansion of regulatory T cells under activation of anti-CD 3 monoclonal antibody and IL-2, which can increase the yield of Treg expansion to a certain extent, but these methods generally have the disadvantages of long time consumption, need of culture for more than 3-4 weeks, harsh culture conditions, high cost and the like, and restrict the application development of regulatory T cells. Therefore, how to effectively increase the production of regulatory T cells has become a hot spot for current research.

Extracellular vesicles are vesicular bodies secreted by cells with a double-layer phospholipid membrane structure, which can carry various bioactive components such as proteins, lipids, nucleic acids, and the like. The target cell is specifically combined by the adhesion molecules and the receptor carried by the surface of the target cell, or the mRNAs, the microRNAs, the proteins and the signal molecules in the content are transferred to the receptor cell, so that the information transfer between cells is mediated. By utilizing the advantage, the extracellular vesicles derived from natural cells or enhanced extracellular vesicles derived from engineered cells are reported to be used for treatment of tumor resistance, transplant rejection resistance, infection resistance and the like, and have wide application prospects.

At present, the separation and purification of extracellular vesicles are generally realized by ultracentrifugation, immunomagnetic beads, reagent precipitation, kit or filter membrane filtration and other methods. For example, publication No. CN108865971A discloses a method and an apparatus for separating exosomes using a porous anodic alumina membrane, but in this method, since a biomaterial that should be separated and extracted remains in a filtration membrane due to an excessively large pore size, a user needs to disassemble and assemble a filtration apparatus for backflushing to obtain the biomaterial, and the outer vesicle may be contaminated during disassembly and assembly, and the extraction purity of the outer vesicle cannot be improved. Or the more common ultracentrifugation method also has the problems of poor separation and extraction purity, poor cell specificity and the like. For example, the patent with application number 201510779110.7 and the patent with application number 201711104768.3 cannot overcome the above defects. In addition, part of methods have the problems of long time consumption, no linear amplification of separation scale, no guarantee of the activity of outer vesicles and high use cost. For example, publication No. CN108865983A discloses a method for extracting extracellular secretion, which still uses centrifugal extraction of polymer precipitate, and the above-mentioned problems are inevitable. Therefore, in order to overcome the drawbacks of the extraction method of extracellular vesicles in the prior art, it is necessary to provide a better method for preparing and separating extracellular vesicles.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to design and provide a preparation method of a genetically engineered regulatory T cell-derived extracellular vesicle and an application technical scheme thereof.

The invention is realized by the following technical modes:

one aspect of the present invention provides a method for preparing a genetically engineered regulatory T cell, comprising the steps of:

s1, obtaining and enriching target cells for infection, wherein the target cells are CD4 ⁺ CD25 ^- Regulatory T cells;

s2, constructing a lentivirus vector, and packaging lentiviruses by utilizing engineering cells to obtain lentivirus particles containing FOXP3 coding genes;

s3, infecting target cells by using the virus obtained in S2, and adding cell stimulating factors to carry out culture screening to obtain a stable cell line for expressing the FOXP3 gene with high purity.

Further, the step of enriching the target cells in S1 specifically comprises the following steps:

obtaining target cell CD4 by using magnetic cell separation system ⁺ CD25 ^- A T cell;

the co-culture was stimulated with anti-CD 3 antibody and soluble anti-CD 28 antibody, plus recombinant IL-2 for 4-6 days.

Further, subcloning the FOXP3 encoding gene into a lentiviral vector in S2 to construct the lentiviral vector containing a reporter element GFP, wherein the primer sequence of the FOXP3 encoding gene is 5; or 5 'CAC TCAAGG AGG CGT TGT C-3',5 'GTT GGC ATA GGT GAA AGG AG-3'.

Furthermore, the engineering cells in S2 are HEK-293T cells.

Further, the S3 specifically includes the following steps:

subjecting the target cells to CD4 ⁺ CD25 ^- T cells were cultured in RPMI1640 medium (containing recombinant IL-2100U/mL, anti-CD 3 antibody 5. Mu.g/mL, anti-CD 28 antibody 5. Mu.g/mL) containing 10% FBS for 24h, the cells were collected and the cell concentration was adjusted to 1.5X 10 ⁶ The RPMI1640 culture medium contains recombinant IL-2100U/mL, 5 mu g/mL of anti-CD 3 antibody and 5 mu g/mL of anti-CD 28 antibody;

in 6-well plates by 1.5X 10 ⁶ Adding the packaged concentrated virus solution into the cell number per well, simultaneously adding polybrene with the final concentration of 8 mu g/mL and 50U/mL recombinant IL-2, continuously culturing for 48h, and collecting transfected cells;

amplifying and maintaining the transfected cells in RPMI1640 medium containing 50U/mL recombinant IL-2 and 10% FBS for 10-14 days, and collecting the transfected cells;

the genetically engineered regulatory T cells were isolated by flow cytometry and resuspended in 10% FBS-containing RPMI1640 medium after centrifugation at 1000r/min for 5min to obtain stable cell lines expressing the specific gene at high purity.

In a second aspect, the present invention provides a method for preparing extracellular vesicles using genetically engineered regulatory T cells prepared as described in any one of the above, comprising the steps of:

1) Collecting a culture solution of high-purity stable cells expressing a specific gene, centrifuging the culture solution, and removing cells and cell debris to obtain a supernatant A;

2) Filtering the supernatant A obtained in the step 1) by using a filter membrane to further remove impurities to obtain a filtrate B;

3) Carrying out size exclusion chromatography on the filtrate B, collecting eluent, and combining the eluent into a certain component according to an elution order to obtain a crude extracellular vesicle solution C;

4) And (3) carrying out rotary ultrafiltration on the extracellular vesicle solution C in the step 3) to obtain an extracellular vesicle solution D.

Further, the parameters of the centrifugal treatment in the step 1) are 4 ℃,2500g and 25-35min; the step 2) adopts a filter for filtration, and the specification of the filter is 0.80 μm.

Further, the solid phase material adopted in the size exclusion method in the step 3) has the following shape: the particle diameter is 40-170 μm, the separation range for globular protein is 10-40000kDa, the separation range for glucan is 10-20000kDa, and the flow rate of the mobile phase is less than 0.1ml/min during size exclusion chromatography.

Further, the parameters for collecting the corresponding components in the step 3) are as follows: collecting from the beginning of elution, wherein the first 42.3-53.9% of the eluent is the 1 st component, and the remaining 46.2-57.7% of the eluent is the 2 nd component, wherein the 1 st component is crude extraction extracellular vesicle solution D.

The third aspect of the invention provides the application of the extracellular vesicles obtained by the preparation method in preparing a medicament for preventing and treating renal transplant rejection, reducing infiltration of inflammatory cells in transplanted kidneys and relieving transplant rejection kinetics, thereby finally improving the long-term survival of renal transplants and the prognosis of transplant recipients.

The beneficial effects of the invention are as follows:

1. the preparation method of the genetically engineered regulatory T cell provided by the invention can overcome the practical problem of low source yield of the existing regulatory T cell, and can obtain the CD4 with simple source and high yield ⁺ CD25 ^- And (3) carrying out T cell transfection by using lentivirus particles so as to obtain a stable cell line for expressing the FOXP3 gene with high purity.

2. By single gene introduction, the gene engineering cell is realized, the problem of multi-gene engineering exogenous gene introduction is solved, and the preparation difficulty and the influence on the cell activity are reduced.

3. The method for preparing and separating the extracellular vesicles can separate vesicle concentrated solution with less protein pollution and high activity, is based on a method combining size exclusion and rotary ultrafiltration, has high separation efficiency and high speed, ensures the separation purity by combining double methods, greatly reduces pollution and reduces the adverse reaction of a user.

4. The method for preparing and separating the extracellular vesicles has small influence on the activity of the extracellular vesicles, can keep higher activity, can effectively reduce the risk of vesicle rupture by lower ultrafiltration pressure, ensures the integrity of the vesicles, and is favorable for improving the effect of treating kidney transplant rejection.

Drawings

FIG. 1 shows CD4 before and after isolation of spleen-derived cells ⁺ CD25 ^- The proportion of T cells;

FIG. 2 is a preparation and characterization of genetically engineered immunoadherent extracellular vesicles (Foe-TEVs);

FIG. 3 is a representation of the biocompatibility of immunoadhesive extracellular vesicles;

FIG. 4 is a graph of genetically engineered immunoadhesion extracellular vesicles (Foe-TEVs) that improve allogeneic rejection kinetics;

FIG. 5 is a depiction of the extensive reduction in inflammatory cell infiltration and macrophage M1-type polarization of transplanted kidney tissue by genetically engineered immunoadhesion extracellular vesicles (Foe-TEVs);

FIG. 6 is a depiction of genetically engineered immunoadhesion extracellular vesicles (Foe-TEVs) to reduce apoptosis in parenchymal cells of transplanted kidneys, alleviate fibrosis, improve renal function, and significantly prolong the survival time of allogeneic transplant recipients.

Detailed Description

The invention is further illustrated by the following specific examples.

Example 1: preparation method of genetically engineered regulatory T cells

S1 obtaining and enriching target cells for infection, wherein the target cells are CD4 ⁺ CD25 ^- T cells, target cells can be derived from spleen or peripheral blood, and are prepared into single cell suspension for separation and acquisition;

wherein: spleen-derived single cell suspensions were obtained by the following steps: firstly, a mouse spleen part sample is required to be obtained, surface bloodstain of the spleen sample is washed by PBS, then grinding and washing are carried out, centrifugation is carried out for 15min at 1500r/min, the centrifugation tube is divided into 4 layers, a milky mononuclear cell layer is taken to obtain mononuclear cells, the mononuclear cells are added into MACS buffer solution, centrifugation is carried out for 110 min 2 times at 1000r/min, the MACS buffer solution is used for resuspending the cells, and counting is carried out for standby application;

a single cell suspension of peripheral blood origin is obtained by the following steps: firstly, 10ml of whole blood is required to be obtained, 10ml of whole blood is transferred into a 50ml centrifuge tube, 10ml of PBS solution is added for dilution, and the mixture is gently mixed; taking two 15ml centrifuge tubes, and adding 5ml of Ficoll solution; slightly adding the diluted blood to the upper layers of Ficoll of two centrifuge tubes, respectively adding 10ml of diluted blood to each centrifuge tube, centrifuging at 2000r/min for 20min to obtain 4 layers in the centrifuge tubes, taking a milky mononuclear cell layer to obtain mononuclear cells, adding PBS to 10-15ml, centrifuging at 1,500r/min for 10min, removing the supernatant, adding MACS buffer solution to resuspend the cells, and counting for later use;

the step of enriching the objective cells comprises isolating CD4 with high purity using a magnetic cell Isolation system Treg Isolation kit (Miltenyi Biotech) ⁺ CD25 ^- T cells, co-culture stimulated with anti-CD 3 antibody (5. Mu.g/mL) and soluble anti-CD 28 antibody (5. Mu.g/mL) plus recombinant IL-2 (100U/mL) for 4-6 days, after 4-6 days of culture expansion, CD4 of said sample relative to the total cell population in said enriched sample ⁺ CD25 ^- T cells reached 97.21% purity (fig. 1A and B).

S2, constructing a lentivirus vector, packaging lentiviruses by utilizing engineering cells to obtain the lentivirus particles containing the FOXP3 coding gene, and specifically comprising the following steps:

in order to prepare a lentiviral expression vector, the mouse FOXP3 gene was subcloned into the self-inactivating lentiviral vector pXZ208, and a plasmid pXZ208-FOXP3-IRES-GFP identified by enzyme digestion was constructed (FIG. 2A);

the primer sequence of the adopted FOXP3 coding gene is 5; or 5 'CAC TCA AGG AGG CGT TGT C-3',5 'GTT GGC ATA GGT GAA AGG AG-3';

packaging lentivirus with 293T cells, and packaging the lentivirus into 4-6X 10 ⁶ The 293T cells of (1) were inoculated into a 100mm plate after polylysine treatment, and 12ml of DMEM complete medium (containing 10% FBS,2 mmol/L-glutamine, no antibiotics) was added, and cultured for 2 days;

transfecting 293T cells with a lentiviral vector encoding the FOXP3 gene, together with 10. Mu.g of packaging plasmid and 5. Mu.g of envelope plasmid, 5% CO ₂ Culturing at 37 deg.C for 24 hr;

after 24h and 48h of transfection, detecting the expression of a reporter gene GFP by using a fluorescence microscope, observing the transfection efficiency, centrifuging at 25000rpm for 2 hours, respectively collecting supernatant, filtering the collected virus supernatant by using a 0.45um filter membrane to obtain slow virus particles containing FOXP3 coding genes, and storing at-80 ℃ for later use;

s3, infecting target cells by using the virus obtained in S2, adding a cell stimulating factor to culture and screen to obtain a stable cell line for expressing the FOXP3 gene at high purity, which specifically comprises the following steps:

subjecting the cells of interest to CD4 ⁺ CD25 ^- T cells were cultured in RPMI1640 medium (containing recombinant IL-2100U/mL, anti-CD 3 antibody 5. Mu.g/mL, anti-CD 28 antibody 5. Mu.g/mL) containing 10% FBS for 24 hours, and the cells were collected and adjusted to a cell concentration of 1.5X 10 ⁶ The RPMI1640 culture medium contains 100U/mL of recombinant IL-2, 5 mu g/mL of anti-CD 3 antibody and 5 mu g/mL of anti-CD 28 antibody;

in 6-well plates at a ratio of 1.5X 10 ⁶ Adding packaged concentrated virus solution into a cell count system, adding polybrene with the final concentration of 8 mu g/mL and 50U/mL recombinant IL-2, continuously culturing for 48h, and collecting transfected cells;

amplifying and maintaining said transfected cells in RPMI1640 medium containing 50U/mL recombinant IL-2 and 10% FBS for 10-14 days, and collecting transfected cells;

flow cytometry isolation of genetically engineered regulatory T cells and centrifugation at 1000r/min for 5min followed by resuspension in RPMI1640 medium containing 10% fbs, obtained stable cell lines expressing FOXP3 gene with purity up to 95.23%.

Example 2: preparation of extracellular vesicles by genetically engineering regulatory T cells

1) Collecting culture solution of stable cells expressing FOXP3 gene with purity as high as 95.23%, centrifuging at 4 deg.C, 2500g,25-35min, and removing cells and cell debris to obtain supernatant A;

2) Filtering the supernatant A obtained in the step 1) by using a filter membrane, wherein the specification of the filter is 0.80 mu m, and further removing impurities to obtain a filtrate B;

3) And (3) carrying out size exclusion chromatography on the filtrate B, collecting eluent, and merging the eluent into a certain component according to an elution sequence to obtain a crude extracellular vesicle solution C, wherein a fixed phase material adopted by the size exclusion chromatography has the following shape: the particle diameter is 40-170 μm, the separation range of the globulin is 10-40000kDa, the separation range of the glucan is 10-20000kDa, and the flow rate of a mobile phase is less than 0.1ml/min during size exclusion chromatography; the corresponding parameters for collecting the corresponding components are: collecting from the beginning of elution, wherein the first 42.3-53.9% of the eluent is a component 1, and the remaining 46.2-57.7% of the eluent is a component 2, wherein the component 1 is crude extraction extracellular vesicle solution D;

4) Adding the extracellular vesicle solution C obtained in the step 3) into a Model 8050 rotary ultrafilter provided with a 100000 NW-CO ultrafiltration membrane for rotary ultrafiltration, switching on nitrogen, controlling the maximum air inlet pressure below 500kPa, and turning on a magnetic stirrer to ensure that the vortex height is 1/3-2/5 of the liquid height. After each ultrafiltration, 60mL of PBS was added and the ultrafiltration was repeated 3 times. After the ultrafiltration, 1mL of PBS was added to resuspend the extracellular vesicles, and an extracellular vesicle solution D was obtained.

Western blot analysis showed that the isolated extracellular vesicles (Vector-TEVs) were highly enriched for classical markers of extracellular vesicles (including CD9, CD63 and TSG 101) compared to cells induced to express the FOXP3 gene. In contrast, extracellular vesicle negative markers (cell-associated markers), including calbindin, histone 3, and GM13, were highly expressed in genetically engineered regulatory T cells, but were barely detectable in their isolated extracellular vesicles and control Vector (Vector) cell-derived extracellular vesicles (fig. 2F). Thus, the above results further support the high purity of the extracellular vesicles isolated under this method. Example 3: characterization of genetically engineered immunoadhesion extracellular vesicles

CD4 induced by FOXP3 gene ⁺ CD25 ^- Synthesis of T cells (Foe-Th cells): 293T cells were transfected with a lentiviral vector encoded by the FOXP3 gene, as shown in FIG. 2A, and then the lentiviral vector containing the FOXP3 gene was introduced into CD4 isolated from spleen with a purity of 95.23% ⁺ CD25 ^- T cells (Th cells). To verify whether Th cells were successfully transfected with the FOXP3 gene, flow cytometry was performed toReporter expression was detected and very high FOXP3 gene-coupled GFP fluorescence intensity was observed in Th cells (fig. 2B). In addition, northern hybridization of FOXP3 mRNA was performed, and it was confirmed that FOXP3 was formed by induction of a lentiviral vector for the FOXP3 gene ⁺ CD4 ⁺ CD25 ⁺ The relative expression of FOXP3 in T cells (Foe-Th cells) increased significantly from 0.11 ± 0.01 (Th cells) to 0.56 ± 0.04 (Foe-Th cells) (fig. 2C), suggesting that the FOXP3 gene was stably transcribed and formed functional mRNA.

Preparation of Foe-Th cell-derived immunoadherent extracellular vesicles (Foe-TEVs): foe-Th cells in culture supernatant were centrifuged at 300g at 4 deg.C (1X 10) ⁷ ) To remove the cells. Then, the cell debris was discarded by centrifugation at 2000g (4 ℃) for 10 minutes. After centrifugation, the supernatant was collected and ultracentrifuged at 100000g for 70 minutes (Avanti J-301, JA 30.50Ti rotor, beckman Coulter, brea, CA, USA) twice to obtain particles containing Foe-TEVs. The particles were washed with 10mL of physiological saline and ultracentrifuged overnight at 100000g (4 ℃). Finally, foe-TEVs (1X 10) were combined ⁹ One) was dispersed in 1mL of PBS and stored at 4 ℃ for 7 days. For long-term storage of Foe-TEVs, storage at-80 ℃ environment may be performed.

Characterization of Foe-Th cell-derived immunoadhesive extracellular vesicles: foe-TEVs isolated from Foe-Th cells were prepared and purified by differential centrifugation and validated for morphology, size and protein markers. Transmission Electron Microscopy (TEM) confirmed that Foe-TEVs had a regular spherical and lipid bilayer structure (FIG. 2D). Nanoparticle Tracking Analysis (NTA) showed that the Foe-TEV diameter was uniformly distributed between 50-500nm with an average diameter of 196.34nm (FIG. 2E). Western blot analysis showed enrichment of EV-associated markers (including CD9 and CD 63) in Vector-TEV and Foe-TEVs, compared to Foe-Th (FIG. 2F). Meanwhile, the presence of the abundant integrins of ITGA4, ITGAL, ITGB1 and ITGB2 in Foe-TEVs was confirmed by Western blot (FIG. 1H). The ELISA method determined the expression levels of functional proteins (TGF-. Beta.1, IL-10 and CTLA-4) in Foe-TEVs. In a word, stable expression of the FOXP3 transcription factor and its target gene product is achieved by selecting exogenous transcripts for genetically engineered Foe-TEVs.

Example 4: biocompatibility testing of genetically engineered adherent extracellular vesicles

To determine the safety and efficacy of Foe-TEVs, biocompatibility studies were performed in vitro by incubating Foe-TEVs or Vector-TEVs with Human Umbilical Vein Endothelial Cells (HUVECs) or renal fibroblasts (KFBs) for 7 days. Cell viability was assessed by live/dead staining and the results showed that cells in the three groups survived and proliferated well with mortality less than 5% (fig. 3A, B, D and E). Notably, the quantitative statistics further supported good biocompatibility for the Foe-TEVs group compared to the control group (fig. 3b, e). Proliferative cytotoxicity is an important parameter for assessing clinical applications in preclinical experimental stages. We then performed the CCK-8 assay to better understand whether Foe-TEVs affect cell proliferation. Over time, cells in all three groups were cultured with similar growth trends, and there was no statistical difference in cell proliferation between the blank control, vector-TEVs, and Foe-TEVs groups on the same day.

Example 5: experiment of kinetics of rejection reaction for alleviating allogeneic body by using genetically engineered immunoadhesion extracellular vesicles

A typical renal rejection reaction model is established by taking Balb/C (H-2 d) mice as transplant donors and C57BL/6 (H-2 b) mice as recipients, and is accompanied by pathological injury mediated by body fluid and cells. In this case, we set up five groups: sham, allograft, vector-TEVs, foe-TEVs and FK506 treated groups (FIG. 4A). We found that the titer of DSA in serum was reduced by 92.38. + -. 8.65% and consistently remained below the positive detection threshold 56 days after Foe-TEVs treatment compared to the Vector-TEVs group (FIG. 4B). Subsequently, we evaluated T cell allograft rejection kinetics after allograft transplantation. After donor splenocyte stimulation, a large number of IFN-. Gamma.secreting T lymphocytes were detected in the Vector-TEVs treated group, indicating a typical cell rejection response to alloantigen stimulation, however, both Foe-TEVs and FK506 inhibited alloreactive T lymphocyte sensitization, with FK506 group showing only 36.37% inhibition, while Foe-TEV treatment decreased to negative levels. (FIG. 4C, D).

B cellActivation of migration in secondary lymphoid organs triggers a Germinal Center (GC) response with maturation of secondary follicles, GL7 ⁺ Activated B cell producing antibody secreting plasma cells (CD 138) ⁺ ) To promote a humoral immune response. Immunofluorescence results show, GL7 ⁺ The follicular proportion was significantly reduced 7 and 21 days after Foe-TEVs treatment, while there was no significant change in the FK506 treatment group (FIG. 4E, G). These data indicate that Foe-TEV with an immunoadhesion effect can achieve a strong and global immunosuppression compared to FK 506. Next, we quantified GC B cells, spleen plasma cells (SPPC) and CD4 ⁺ Th cells were evaluated for the extent of antibody-mediated rejection (fig. 4F). CLT 21 days post-transplantation, post Foe-TEVs treatment ⁺ FAS ⁺ 、CD138 ⁺ SPPC and CD4 ⁺ Infiltration rates of Th cells were significantly lower than both FK506 and Vector-TEVs treated groups (fig. 4H-J). These results demonstrate that Foe-TEV effectively inhibits the alloreactive T cell response and B cell activation in secondary lymphoid organs by immunoadhesion-mediated contact and non-contact immunomodulation, significantly reduces the allosensitization response, and further demonstrates the potential of immunoadhesive Foe-TEV for use in transplant rejection.

Example 6: experiment for inhibiting inflammatory cell infiltration of transplanted kidney tissue and macrophage M1 type polarization by using extracellular vesicle of genetically engineered immunoadhesion

We examined IFN- γ deposition in graft tissue by immunohistochemistry and Foe-TEVs treatment was effective in reducing IFN- γ deposition from 16.94 + -1.76 to 12.59 + -1.31 (FIG. 5A, C) compared to controls. Immunohistochemistry showed that Foe-TEVs also significantly reduced the deposition of C4d (FIG. 5B, D). At the same time, we assessed the recruitment of immune cells in the graft by immunofluorescence staining (fig. 5E). Studies have shown that FK560 is responsible for CD3 in allografts ⁺ Infiltration of T cells had some inhibition, but inhibition of Foe-TEV was more pronounced (fig. 5F). Furthermore, foe-TEVs treatment significantly reduced CD11c compared to FK560 ⁺ DC and F4/80 ⁺ Infiltration of macrophages, a unique therapeutic effect of Foe-TEVs (FIG. 5G, H). Furthermore, flow cytometry analysis results show that the immune adhesionThe additional Foe-TEVs significantly increased CD4 in the graft ⁺ FOXP3 ⁺ Proportion of Treg cells (fig. 5I).

Macrophages are abundant in allografts and are closely associated with the development of graft rejection. We examined the number and distribution of M1-type macrophages. Intra-graft p-STAT-1 following treatment with Foe-TEVs ⁺ The infiltration ratio of macrophages was reduced from 17.56 + -2.31% to 2.45 + -0.31% (FIG. 5J, K). Furthermore, the percent infiltration of M1-type macrophages decreased gradually over time, with the percent infiltration being significantly lower in the Foe-TEVs treated group than in the Vector-TEVs group on both day 14 and day 28.

Taken together, foe-TEVs with contact-dependent and independent effects of immunoadhesion alleviate allograft rejection from multiple levels by effectively inhibiting IFN- γ and C4d deposition, reducing alloreactive immune cell infiltration and macrophage M1-type polarization, while increasing the proportion of Treg cells.

Example 7: genetically engineered immunoadhesion extracellular vesicles (Foe-TEVs) reduce apoptosis of transplanted kidney parenchyma, relieve fibrosis, improve renal function, and significantly prolong the survival time of allogeneic transplant recipients.

We performed structural and functional analysis of allograft kidneys in recipient mice. Most of the glomeruli and tubules remained in normal structures after treatment with Foe-TEVs, as shown by HE staining and PAS staining, similar to the sham group (FIG. 6A). Also, apoptotic cells were significantly reduced after Foe-TEVs treatment compared to the Vector-TEVs group and the FK506 group (FIGS. 6D-F). The index of interstitial fibrosis of the grafts (0.43. + -. 0.02) was found to be reduced by Masson staining at 8 weeks post-transplantation in the Foe-TEV treated group compared to Vector-TEVs (2.39. + -. 0.18). Also, tubular atrophy and glomerulosclerosis were significantly reduced following treatment with Foe TEVs (FIGS. 6H-K). The renal function indices showed that the Glomerular Filtration Rate (GFR) was increased more than 2-fold at

weeks

4 and 8 after Foe-TEV treatment compared to Vector-TEVs treatment, while the urinary protein/creatinine ratio was significantly reduced, whereas no significant difference was observed compared to sham and allograft mice (fig. 6b, c). At 12 weeks, foe-TEVs treatment significantly improved survival of mice (83.56%) (FIG. 6L). In conclusion, foe-TEVs treatment has a reversal effect on transplanted kidney fibrosis, remarkably improves the transplanted kidney function, prolongs the survival period of a kidney transplant receptor (from 30.16 days to 92.81 days), and provides a promising treatment method for kidney transplant rejection resistance treatment.

Claims

1. A method for preparing a genetically engineered regulatory T cell comprising the steps of:

s1, obtaining and enriching target cells for infection, wherein the target cells are CD4 ⁺ CD25 ^- A T cell;

s2, constructing a lentiviral vector, and packaging lentiviruses by using engineering cells to obtain lentivirus particles containing FOXP3 coding genes;

s3, infecting target cells by the lentivirus obtained in S2, and adding cell stimulating factors to culture and screen to obtain a stable cell line for expressing the FOXP3 gene in high purity.

2. The method of claim 1, wherein the step of enriching S1 for desired cells comprises the steps of:

the anti-CD 3 antibody, the soluble anti-CD 28 antibody and the recombinant IL-2 are added into the culture medium to maintain the culture for 4 to 6 days.

3. The method of claim 1, wherein the FOXP3 encoding gene is subcloned into a lentiviral vector in S2 to construct a lentiviral vector containing a reporter element GFP, and the primer sequence of the FOXP3 encoding gene is 5; or 5 'CAC TCA AGG AGG CGT TGT C-3',5 'GTT GGC ATA GGT GAA AGG AG-3'.

4. The method of claim 1, wherein the engineered T cell in S2 is a HEK-293T cell.

5. The method of claim 1, wherein S3 comprises the steps of:

subjecting the cells of interest to CD4 ⁺ CD25 ^- T cells were cultured in 10-vol FBS-containing RPMI1640 medium for 24 hours, and the cells were collected and adjusted to a cell concentration of 1.5X 10 ⁶ The RPMI1640 culture medium contains recombinant IL-2100U/mL, 5 mu g/mL of anti-CD 3 antibody and 5 mu g/mL of anti-CD 28 antibody;

in 6-well plates by 1.5X 10 ⁶ Adding packaged concentrated virus solution into the cell number per well, adding polybrene with final concentration of 8 μ g/mL and 50U/mL recombinant IL-2, continuously culturing for 48h, and collecting transfected cells;

6. A method for preparing extracellular vesicles using the genetically engineered regulatory T-cells prepared according to any one of claims 1 to 5, comprising the steps of:

2) Filtering the supernatant A in the step 1) by using a filter membrane to further remove impurities to obtain a filtrate B;

7. The method according to claim 6, wherein the centrifugation in the step 1) is carried out at 4 ℃ at 2500g for 25-35min; and 2) filtering by adopting a filter in the step 2), wherein the specification of the filter is 0.80 mu m.

8. The method of claim 6, wherein the size exclusion method of step 3) uses a stationary phase material having the following shape: the particle diameter is 40-170 μm, the separation range for globular protein is 10-40000kDa, the separation range for glucan is 10-20000kDa, and the flow rate of the mobile phase is less than 0.1ml/min during size exclusion chromatography.

9. The method according to claim 6, wherein the parameters for collecting the corresponding components in step 3) are: collecting from the beginning of elution, wherein the first 42.3-53.9% of the eluent is the 1 st component, and the remaining 46.2-57.7% of the eluent is the 2 nd component, wherein the 1 st component is crude extraction extracellular vesicle solution D.

10. Use of the extracellular vesicles obtained by the method of any one of claims 6 to 9 for the preparation of a medicament for the prevention and treatment of chronic rejection in renal transplantation, for the reduction of inflammatory cell infiltration in transplanted kidneys, or for improving renal transplantation survival.