Application of melanoma-derived exosome in medicine for preventing and treating corneal transplantation immunological rejection

Abstract

The invention provides an application of a melanoma-derived exosome in a medicine for preventing and treating corneal transplantation immunological rejection, belongs to the technical field of ophthalmology biological medicines, and adopts a mouse corneal transplantation model to find that B16-Exo can inhibit immunological rejection, which is represented by corneal opacity, neovascularization, alleviation of immune disorder and improvement of postoperative survival rate. Proteome analysis reveals differential expression of key protein in B16-Exo, especially JAK2 protein in a JAK-STAT signal channel, further mechanism research clarifies the effect of the JAK2 protein in enhancement of myeloid-derived suppressor cell activity and inhibition of T cell proliferation, and these findings prove that B16-Exo has a remarkable immunoregulation effect in transplantation immunology, and can be used for preparing a medicine for treating myeloid-derived suppressor cell activity and T cell proliferation. And a basis is provided for further exploring the tumor cell exosome as an effective therapeutic agent for managing immunological rejection in organ transplantation.

Description

Technical Field

[0001] The present invention belongs to the technical field of ophthalmic biomedicine, and specifically relates to the application of melanoma-derived exosomes in a drug for preventing and treating corneal transplant immune rejection. Background Art

[0002] In organ and tissue transplantation, effective management of immune rejection is a huge challenge, which mainly involves the recipient's immune response to the allogeneic graft. Post-transplant immune rejection triggers a multifaceted immune response cascade, in which host immune cells recognize and initiate responses against xenoantigens in the transplanted tissue. T cells play a central role in attacking transplanted tissues and coordinating other immune responses, exacerbating tissue damage. Therefore, effective management of immune rejection is crucial to improving transplant outcomes. Currently, the management of immune rejection mainly relies on long-term use of immunosuppressants. Although these drug therapies significantly limit immune rejection, they also bring a series of side effects, including increased susceptibility to infection, metabolic disorders, and neurological complications. Therefore, the development of new immune regulation strategies has great potential in improving the clinical efficacy of organ or tissue transplantation. These strategies need to not only effectively alleviate immune rejection, but also improve long-term graft survival, thereby providing patients with safer treatment outcomes and broadening the horizons of transplantation therapy.

[0003] In recent years, exosomes have attracted attention as promising candidates for immunomodulation. Exosomes are nanoscale vesicles released by cells that carry bioactive molecules such as proteins, lipids, and nucleic acids, facilitating intercellular signaling within the immune system. Indeed, specific subsets of exosomes have been shown to modulate immune cell function, cytokine dynamics, and immune responses, and have demonstrated their therapeutic potential in a variety of immune-related pathological conditions. The immunomodulatory properties of exosomes show context-dependent variability depending on their cellular source and application. The immunomodulatory activity of exosomes has been studied in the management of corneal transplants. For example, exosomes derived from human induced pluripotent stem cells, bone marrow mesenchymal stem cells, human adipose tissue, and umbilical cord blood stem cells have shown potential to combat corneal transplant rejection.

[0004] However, the high cost limits its widespread application. Although exosomes from other immune cells such as MDSCs and dendritic cells (DCs) also exhibit immunomodulatory effects, their effects tend to promote immune responses or support tissue repair rather than direct immunosuppression. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an application of melanoma-derived exosomes in a drug for preventing and treating corneal transplant immune rejection, using exosomes from B16-F10 melanoma cells (B16-Exo) as an example to illustrate the therapeutic potential of tumor cell exosomes in alleviating transplant immune rejection, and using mouse corneal transplantation as a model system to confirm the immunosuppressive effect of B16-Exo after transplant rejection, accompanied by an increase in postoperative survival rate. The unique transparency and avascularity of the cornea make it an ideal experimental platform for studying immune rejection, neovascularization and its regulatory process.

[0006] To achieve the above objectives, this protocol first provides the use of melanoma-derived exosomes in drugs for preventing and treating corneal transplant immune rejection.

[0007] Preferably, the method for preparing melanoma-derived exosomes is as follows: exosomes are isolated from the culture supernatant of a melanoma cell line B16-F10, the cells are cultured in Dulbecco's modified Eagle's medium, supplemented with 10% exosome-removed fetal bovine serum, penicillin and streptomycin, the collected supernatant is centrifuged at 2000xg and 10000xg, filtered, large vesicles, smaller apoptotic bodies and microvesicles are removed in turn, and then ultracentrifuged twice at 100,000xg at 4°C to obtain exosomes.

[0008] Preferably, the centrifugation times at 2000 x g and 10000 x g are 10 min and 60 min respectively.

[0009] Preferably, the ultracentrifugation at 100,000 x g is performed twice for 90 min.

[0010] Preferably, the effective dose of the melanoma-derived exosomes in the drug is 1 μg.

[0011] Preferably, the dosage form of the drug includes an injection.

[0012] The mechanism of B16-Exo in the prevention and treatment of corneal transplant immune rejection is:

[0013] like Figure 1As shown, B16-Exo persisted in the corneal microenvironment for more than 6 days, accompanied by significant reduction in corneal opacity, neovascularization and immune infiltration. Proteomic analysis and mechanism elucidation demonstrated the immunomodulatory properties of B16-Exo, which mediated by JAK2 protein components, regulated the JAK-STAT signaling axis, enhanced the proliferation and immunosuppressive ability of myeloid-derived suppressor cells (MDSCs), and effectively regulated the proliferation dynamics of T cells. The immunomodulatory properties of B16-Exo included reducing corneal opacity, neovascularization and immune infiltration, and improving postoperative survival rate. Both in vitro and in vivo experiments revealed the key role of the JAK-STAT signaling pathway in inhibiting B16-Exo-mediated corneal transplant rejection.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) This study discovered for the first time that B16-Exo has a therapeutic effect in inhibiting corneal transplant immune rejection, providing a new strategy for the development of immunomodulators in organ transplant management.

[0016] (2) Tumor-derived exosomes mimic and apply tumor immunomodulatory mechanisms in the ocular microenvironment, thereby effectively inhibiting the immune response. This groundbreaking discovery lays the foundation for the use of tumor cell-derived exosomes to manage ocular transplant rejection.

[0017] (3) B16-Exo appears to be a promising therapeutic agent that can maintain corneal transparency, regulate local immunity, and prolong the survival of allogeneic corneal transplants, highlighting its potential application value in transplant medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The mechanism of B16-Exo in the prevention and treatment of corneal transplantation immune rejection;

[0020] Figure 2The therapeutic effect of B16-Exo in reducing immune rejection in allogeneic corneal transplantation in Experimental Example 1. (A) Transmission electron microscopy images of B16-Exo and JB6-Exo and their particle size distribution, scale bar: 100 nm, (B) Western blot analysis of exosome markers Alix, CD63, and GAPDH in different exosomes, (C) Tracking of Dio-labeled exosomes injected into mice receiving corneal transplantation by subconjunctival injection, scale bar: 5 00 μm (original image), 100 μm (enlarged image), (D) schematic diagram of corneal transplantation, (E) Kaplan-Meier survival curves of allogeneic transplantation and allogeneic transplantation with indicated administration (n=6 / group), (F) Slim-lamp image of cornea with transplantation on day 18, (G) HE staining of corneal transplantation on day 18 after transplantation, scale bar: 200 μm, (H) immune cells (CD45 + ) and T cells (CD3 + ) staining. Scale bar: 100 μm, (I) CD45 in the whole corneal graft + The percentage of immune cells, (J) CD3 + Percentage of T cells, bars in (I) and (J) represent mean ± SD (n = 3);

[0021] Figure 3 B16-Exo inhibits angiogenesis in Experimental Example 2. (A) is an image processing for evaluating neovascularization in a cornea containing a transplant. The cornea containing a transplant is encircled by a dotted circle in the left figure (left), and the entire corneal area is captured (middle). The right figure shows the neovascularization area after treatment. (B) is a representative immunofluorescence micrograph of CD31 staining (for new blood vessels) of a cornea containing a transplant on day 18 after transplantation. (C) is a representative immunofluorescence micrograph from Figure 2 B is the quantitative analysis of the neovascularization area, (D) is the representative image of the tubular structure formation of HUVECs treated with VEGF combined with JB6-Exo or B16-Exo, and (E) is the quantitative analysis of the tubular structure formation;

[0022] Figure 3 B16-Exo regulates the proliferation and function of myeloid-derived suppressor cells in Experimental Example 3. (FH) are the percentage of myeloid-derived suppressor cells (MDSCs), multinuclear myeloid-derived suppressor cells (PMN-MDSCs) and mononuclear myeloid-derived suppressor cells (M-MDSCs) in the blood, respectively. + The normalized percentage of immune cells (IK) is the percentage of MDSCs, PMN-MDSCs and M-MDSCs in the spleen, respectively. +Normalized percentage of immune cells, (L) quantification of serum IL-6 levels on day 18 after transplantation, the bar graphs in the figure represent the mean ± standard deviation (n = 3 / group);

[0023] Figure 4 The differential expression and potential functional significance of JAK2 in B16-Exo in Experimental Example 4, (A) is a Venn diagram showing the number of common proteins in B16-Exo and JB6-Exo, (B) is a volcano plot of the distribution of differentially expressed proteins, (C) is a protein-protein interaction (PPI) network, (D) is a KEGG functional enrichment analysis of B16-Exo using gene set enrichment analysis (GSEA), (E) is a heat map showing differentially expressed proteins in the JAK-STAT signaling pathway based on GSEA, (F) is a Western blot validation of the expression levels of JAK2 and AKT1 proteins, and (G) is the enrichment score (ES) of the JAK-STAT signaling pathway;

[0024] Figure 5 Figure 5 shows that B16-Exo mediates the expansion and function of MDSCs through the JAK2 pathway. (A) is a representative flow cytometry image of MDSCs. (B) is the number of MDSCs, (C) PMN-MDSCs, and (D) M-MDSCs in the presence of MDSC polarization medium (IL-6 and GM-CSF) after various treatments. (E) is the number of MDSCs collected and treated with the indicated exosomes for 72 hours in the absence or presence of anti-JAK2, and compared with CD4 + T cells were co-cultured for 5 days. (F) CFSE-labeled CD4 + T cell proliferation;

[0025] Figure 6 The role of JAK2 in B16-Exo-induced MDSC expansion and function in Experimental Example 6, (A) is the survival curve after transplantation after different treatments, (B) is the corneal surface image, (C) is the corneal cross-sectional image stained with HE, (D) is the immunofluorescence staining image showing the CD45 and CD3 markers at the junction of the transplant host (left) and the transplant center (right), (EF) are the quantitative analysis of CD45 and CD3 immune cells, (G) is the corneal image of fluorescently labeled blood vessels, (H) is the quantitative analysis result of the neovascularization area, and (IN) are the proportion and distribution of MDSC and its subtypes in the spleen and blood, respectively;

[0026] Figure 7 The effect of B16-Exo on MDSC activity was evaluated by CCK-8 assay in Experimental Example 3;

[0027] Figure 8 MDSC (CD11b +Gr-1 + )、PMN-MDSC(CD11b + Gr-1 + 6G + Ly6Clo) and M-MDSC (CD11b + Gr-1 + Representative flow cytometry images of Ly6G-Ly6Chi cells;

[0028] Fig. 9 Quantitative analysis of Treg populations in mouse tissues after transplantation in Experimental Example 3. (A) Flow cytometry gating strategy used to identify CD45 in mouse tissues on day 18 after transplantation. + cells, CD4 + T cells and Tregs (CD4 + CD25 + FoxP3 + ), (BE) are quantitative analysis of Treg populations in spleen (B), blood (C), bone marrow (D) and cervical lymph nodes (E), respectively. DETAILED DESCRIPTION

[0029] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0030] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0031] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the examples are commercially available.

[0032] Example 1

[0033] Isolation and characterization of exosomes

[0034] Exosomes were isolated from the culture supernatant of mouse melanoma cell line B16-F10 and mouse epidermal cell JB6 Cl41. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, MA, USA) supplemented with 10% exosome-depleted fetal bovine serum (Exo-FBS, System Biosciences, CA, USA), 100 U / mL penicillin, and 100 μg / mL streptomycin (Thermo Fisher Scientific, MA, USA). The collected supernatant was centrifuged at 2000 x g for 10 min; 10000 x g for 60 min, and filtered through a 0.22 μm pore filter (MF-Millipore) to remove large vesicles, smaller apoptotic bodies, and microvesicles in turn. The exosomes were then ultracentrifuged twice at 100,000 x g for 90 min at 4 °C to precipitate the exosomes and gently resuspended in 1x PBS.

[0035] Example 2

[0036] Exosome tracking and distribution

[0037] To track the distribution of exosomes throughout the eye after subconjunctival injection (SCI), exosomes were labeled with Dio (green fluorescence, Shanghai Sangon Biotech). Exosomes were first diluted in 1 ml PBS and labeled with 5 ul Dio (2x10 6 M) staining for 20 minutes at room temperature, and unbound Dio was removed by ultracentrifugation at 100,000xg for 60 minutes. Dio-labeled exosomes were injected subconjunctivally to track the distribution in the ocular tissues carried by the transplant. On the 6th day after transplantation, the entire eyeball with the conjunctiva was removed, and OCT-embedded frozen sections (10 μm) were made and mounted with Fluoroshield histological mounting solution containing DAPI (Sigma-Aldrich, St. Louis, USA).

[0038] Example 3

[0039] (1) Mice and corneal transplantation

[0040] All mice used in this experiment were housed in a specific pathogen free facility. Male BALB / C and C57BL / 6 mice, aged six to eight weeks, were purchased from Slake Jingda Laboratory Animal Co., Ltd., Hunan, China, and used for corneal transplantation. For allogeneic and homologous transplantation procedures, BALB / C mice served as recipients, while both C57BL / 6 and BALB / C served as donors.

[0041] After donor mice were sacrificed by cervical dislocation, a 2.25 mm diameter full thickness corneal section was carefully excised. Before receiving the transplant, the recipient mice were anesthetized by intraperitoneal injection of 16.5 mg / ml sodium pentobarbital. The corneal grafts from the donor were then sutured to the recipient's 2 mm diameter graft bed using 11-0 nylon sutures and needles. The tarsorrhaphy was performed immediately after the surgery using 8-0 nylon sutures and needles. Postoperative care included the removal of the sutures of the palpebral and cornea on the 3rd and 7th days after surgery, respectively. All graft conditions were monitored and evaluated every other day from the 7th day onwards by a slit lamp biomicroscope system (WeztlarLeicca, Germany). The observers were blinded to the experimental design. When the grafts showed corneal stroma opacity (i.e., the iris texture and pupil margin were not visible), they were considered rejected. All animal experiments were performed in accordance with the animal ethics guidelines of the Experimental Animal Center of Central South University and approved by the Center (File No.: 20231195).

[0042] (2) The experimental operation is as follows:

[0043] 2.1 Quantitative analysis of corneal neovascularization

[0044] Quantitative analysis of new angiogenesis in corneal grafts was performed by immunofluorescence staining to evaluate the appearance of new blood vessels. On the 18th day after transplantation, the cornea carrying the graft was removed from the eyeball and fixed in acetone for 20 minutes. In order to reduce nonspecific binding, the fixed cornea was blocked with PBS containing 10% donkey serum (Absin Bioscience, Shanghai) for 1 hour. In order to identify corneal blood vessels, the tissue was incubated overnight at 4°C with a specific primary antibody for CD31 (R&D Systems, Minnesota, USA), and then incubated with a specific secondary antibody Alexa-Flour 594 (Jackson, Pennsylvania, USA) at room temperature for 2 hours. After staining, the cornea was loaded with Fluoroshield histological mounting medium containing DAPI (Sigma-Aldrich, St. Louis, USA) to maintain fluorescence and nuclear staining. The loaded samples were stored in the dark at 4°C until imaging. Image J (NIH Image J System, Version 1.8.0, Bethesda, USA) was used to quantify the percentage of new vascular perfusion area of ​​the entire cornea.

[0045] 2.2 In vitro angiogenesis

[0046] First, place the matrix gel in a 4°C refrigerator overnight to melt, and pre-cool the 24-well plate, 200μl pipette tip and 1.5ml EP tube to -20°C for use. The next day, take the matrix gel out of the refrigerator and quickly dispense it into the pre-cooled EP tube. All operations are completed on the ice box to avoid premature solidification of the matrix gel. Using a pre-cooled pipette tip, draw 200μl of matrix gel from the EP tube and evenly add it to each well of the 24-well plate. Be careful to avoid bubbles during the process. After laying, place the 24-well plate in a cell culture incubator at 37°C and wait for 30 to 45 minutes until the matrix gel is completely solidified. During this period, prepare a single cell suspension of vascular endothelial cells and count them. When the matrix gel solidifies, adjust it to 1×10 5 The cell suspension with a density of 100 cells / mL was evenly added to each well, and the tip of the pipette was kept vertical and did not touch the bottom of the well. After the inoculation, the cells were cultured for 16 hours at 37°C and 5% CO2. After the culture, angiogenesis was observed and recorded using an optical inverted microscope, and the length of new blood vessels was counted in 5 randomly selected fields to evaluate the angiogenesis effect.

[0047] 2.3 Histology and immunohistochemistry

[0048] Hematoxylin and eosin (H&E) staining was performed to determine corneal irregularity, corneal edema, and the degree of inflammatory cell infiltration. + and CD3 + Cell infiltration staining indicates the presence of immune cells and T cells, respectively. On day 18 after transplantation, whole eyeballs carrying grafts were fixed with 4% formaldehyde and then embedded in paraffin in preparation for H&E and immunohistochemical staining.

[0049] Paraffin-embedded sections were first dewaxed and dehydrated. The sections were then placed in a repair box filled with EDTA antigen retrieval buffer (Servicebio, Wuhan, China) and the antigens were repaired in a microwave oven. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 15 minutes, and then the protein was blocked in serum blocking solution (10% goat serum, 1% bovine serum albumin, 22 mg / ml glycine, 0.1% Tween-20) for 1 hour. Incubate with primary antibody (Abcam Bioscience, Cambridge, USA) at 4°C overnight, followed by incubation with HRP secondary antibody (AiFang Biology, Hunan, China) at room temperature in the dark for 40 minutes. Fluorescent dyes TYR-570 and TYR-520 were used for CD45, respectively. + and CD3 + Visualization of cells. Slides were mounted with Fluoroshield Histology Mounting Solution containing DAPI (Sigma-Aldrich, St. Louis, USA) and photographed under a microscope (Axio Imager M2, Carl Zeiss, Oberkochen, Germany). +Apotome 3).

[0050] 2.4 Bone marrow cell and MDSC isolation

[0051] Bone marrow cells were isolated from the bone marrow of Balb / c mice and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS; Gibco, Australia), 100 units / mL of penicillin, and 100 μg / mL of streptomycin. In order to specifically stimulate the proliferation of myeloid-derived suppressor cells (MDSCs) in bone marrow cell culture, 10 ng / mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng / mL of interleukin 6 (IL-6) (Perprotech, Rocky Hill, USA) were added to the culture medium.

[0052] 2.5 Flow cytometry quantification

[0053] Flow cytometry was used to assess the level of MDSC in mouse tissues after transplantation or in vitro MDSC culture. Single cell suspensions were obtained from bone marrow, blood, and spleen on day 18 after transplantation. Dead cells were stained with ZombieAqua fixable viability dye (BioLegend, CA, USA) after destruction of erythrocytes (BD, NJ, USA). All antibodies were purchased from BD Biosciences, NJ, USA. Fc receptors were blocked using anti-CD16 / 32 antibodies before all surface staining. MDSCs were then identified using APC-Cy7CD45 antibodies, APC CD11b antibodies, and PE granulocyte differentiation antigen 1 (Gr-1) antibodies. FITC lymphocyte antigen 6 complex, gene C (Ly6C) antibodies and PerCP-Cy5.5 lymphocyte antigen 6 complex, gene G (Ly6G) antibodies were used to identify MDSC subsets, namely mononuclear MDSC (M-MDSC) and polymorphonuclear MDSC (PMN-MDSC). Total MDSC, M-MDSC, and G-MDSC were identified by CD45, respectively. + CD11b + Gr-1 + 、CD45 + CD11b + Gr-1 + Ly6G-Ly6Chi and CD45 + CD11b + Gr-1 + 6G + Ly6Clo cell population identification.

[0054] 2.6 Cytokine Quantification

[0055] Cytokine (IL-6, IL-2, IL-4, IFN-γ, TNF, IL-17A, and IL-10) levels in mouse serum were measured using the BD Cytokine Bead Array Kit (BD, New Jersey, USA). The manufacturer's instructions were followed. After capturing the cytokine molecules present in the serum with beads coated with specific antibodies, the bound beads were detected and quantified using a Northern Lights flow cytometer (CYTEK NL-3000, USA).

[0056] 2.7 Proteomic analysis of exosomes

[0057] The exosome samples were mixed with an appropriate amount of lysis buffer, heated in a boiling water bath for 15 minutes, and then centrifuged at 14,000g for 15 minutes. The supernatant was collected. The protein concentration was quantified using the BCA method and stored at -80°C. 20 μg of protein was taken from each sample, 6X loading buffer was added, and the samples were heated in a boiling water bath for 5 minutes, followed by 12% SDS-PAGE electrophoresis at a constant voltage of 250V for 40 minutes, and the gel was stained with Coomassie Brilliant Blue. For mass spectrometry preparation, 100 μg of protein solution in each sample was treated with TCEP to a final concentration of 10 mM and heated again for 10 minutes, then cooled to room temperature. CAA was added to a final concentration of 40 mM and incubated at room temperature for 30 minutes in the dark. Then, 100 μL of 50 mM NH4HCO3 solution and 40 μL of trypsin buffer (4 μg trypsin in 40 μL 50 mM NH4HCO3 solution) were added, shaken at 600 rpm for 1 minute, and incubated at 37°C for 16-18 hours. Then, an appropriate amount of 20% TFA was added to make the final concentration reach 0.5%, and centrifuged again at 14,000g for 15 minutes. The supernatant was taken and desalted using a C18 column. The desalted peptides were lyophilized, re-dissolved with 40 μL of 0.1% formic acid solution, and the peptide concentration was determined by OD280. The samples were separated at a nanoliter flow rate in a NanoElute system and connected to a timsTOF Pro mass spectrometer equipped with a CaptiveSpray ion source (Bruker, Bremen, Germany). Buffer A was 0.1% formic acid in water, and buffer B was acetonitrile in water containing 0.1% formic acid (acetonitrile concentration was 100%). The column was balanced with 100% A solution, and the sample was loaded onto the analytical column (IonOpticks, Australia, 25 cm x 75 μm, C18 filler 1.6 μm) by an autosampler at a flow rate of 300 nL / min. After chromatographic separation, the sample was analyzed by the PASEF mode of the timsTOF Pro mass spectrometer. The ionization mode was set to positive ion, the parent ion scan range was 100-1700 m / z, and the ion mobility range was 0.75-1.4 V·s / cm 2The ion accumulation or release time was 100 ms, the ion utilization was 100%, the capillary voltage was set to 1500 V, the drying gas flow rate was 3 L / min, and the drying temperature was 180 °C. The PASEF settings were: 10 MS / MS scans per cycle (total cycle time of 1.16 s), charge range of 0–5, dynamic exclusion time of 0.5 min, ion target intensity of 10,000, ion intensity threshold of 2500, and collision-induced dissociation (CID) fragmentation energy range.

[0058] 2.8CD4 + T cell proliferation assay

[0059] According to their inhibition of CD4 + The ability of MDSC to proliferate T cells was used to evaluate the immunosuppressive capacity of MDSC. CD4 + T cells were stained with CD4 (L3T4) microbeads (Miltenyi Biotec). CD4 was then prestained with carboxyfluorescein succinimidyl ester (2.5 uM, Invitrogen, California, USA). + T cells. Cells were cultured in RPMI-1640 (Gibco, Shanghai, China) supplemented with 10% fetal bovine serum, 1% HEPES buffer (LIFE technologist, New Mexico, USA), 1% sodium pyruvate (LIFE technologist, New Mexico, USA), and 1% non-essential amino acids (LIFE technologist, New Mexico, USA). CD4+ T cells were stimulated using culture plates pre-coated with 5 μg / ml anti-CD3 and 2 μg / ml anti-CD28 antibodies. Equal numbers of MDSCs were mixed with CD4 + T cells (1x10 5 / well) were co-cultured in 96-well round button plates (Corning, New York, USA). After 5 days, CD4 was further stained with PE-conjugated CD4 antibody + T cells. The proliferation rate was detected using Northern Lights flow cytometer.

[0060] 2.9 Statistical analysis

[0061] Data collected in this study were statistically analyzed using SPSS software, version 20 (SPSS Inc., Chicago, USA). Corneal graft survival was plotted according to the Kaplan-Meier method and compared by log-rank test. Analysis of variance (ANOVA) and Dunnett's test were used for multiple group comparisons. Means of two groups were compared using independent t-tests. All data were visualized using GraphPad Prism 8.4.0 (San Diego, CA, USA).

[0062] Experimental Example 1

[0063] To investigate the therapeutic effect of B16-Exo in reducing immune rejection in allogeneic corneal transplantation

[0064] The exosomes (B16-Exo) from B16-F10 melanoma cells isolated in Example 1 were used as an experimental model, and the exosomes (JB6-Exo) from JB6 Cl41 normal epidermal cells were used as a control. B16-Exo and JB6-Exo were separated from the culture supernatants of B16-F10 and JB6 Cl41 cells by gradient centrifugation.

[0065] Finally, the exosomes were quantified using the BCA protein assay kit from Vazyme Biotech (Nanjing, China). Exosomes were observed by electron microscopy (Hitachi HT7800, Tokyo, Japan), and their size distribution was determined using a nanoparticle size tracking analyzer (Malvern Instruments, UK). The expression of exosome markers Alix, CD63, and negative marker GAPDH (Proteintech, Chicago, USA) was tested using Western blot analysis.

[0066] Transmission electron microscopy (TEM) examination showed that these exosomes had a typical spherical morphology ( Figure 2 A), the size distribution is mainly around 100 nm, which is consistent with the dynamic light scattering (DLS) measurement results. WB analysis further confirmed the presence of exosome markers CD63 and Alix ( Figure 2 B), verifying the successful isolation of exosomes.

[0067] To evaluate the distribution and persistence of exosomes in the ocular region, 1 μg of fluorescently labeled (Dio) B16-Exo and JB6-Exo were injected into allogeneic mice by subconjunctival injection (SCI). Fluorescent signals could still be observed in the cornea and conjunctiva 6 days after injection ( Figure 2 C), indicating that exosomes have the ability to remain in ocular tissues for a long time. This property not only suggests the possibility of reducing the frequency of in vivo administration, but also may increase patient compliance. Therefore, subsequent studies adopted a dosing regimen of once-weekly injections.

[0068] Further studies aim to evaluate the therapeutic effects of these two exosomes in a mouse allogeneic corneal transplantation model, e.g. Figure 2D is a schematic diagram of corneal transplantation, which was performed on day 0, and exosomes or PBS were co-administered once a week (1 μg exosomes) until day 35. Corneas were observed every other day until day 42. Mice were randomly selected and sacrificed on day 18 for hematoxylin-eosin (HE) staining, immunofluorescence staining (IF), and flow cytometry (FCM).

[0069] Untreated allogeneic mice showed graft rejection and died before the designated observation endpoint, with an average survival of 15 ± 1.0 days. Administration of JB6-Exo showed a modest effect, with death occurring after the second dose (day 14) and a survival rate of only 20% within 42 days. In sharp contrast, treatment with B16-Exo significantly prolonged the survival of mouse grafts, with a survival rate of 70% within 42 days ( Figure 2 E). Notably, corneal transparency, a key indicator of transplantation success, was significantly improved in the B16-Exo group compared with the control group on day 18 ( Figure 2 F). H&E staining of the corneal stroma further confirmed the significant reduction of corneal stromal edema in the B16-Exo group, which was restored to a level similar to that of allogeneic mice ( Figure 2 G).

[0070] Given that corneal allograft rejection involves an inflammatory imbalance caused by excessive immune system activation, the expression of immune cells (CD45 + ) and T cells (CD3 + The results showed that B16-Exo treatment significantly reduced the infiltration of these immune cells ( Figure 2 H), the fluorescence intensity was similar to that of the allogeneic control, emphasizing the inhibitory effect of B16-Exo on immune cell infiltration. In contrast, mice treated with JB6-Exo showed obvious immune cell infiltration, indicating weak immunomodulatory capacity. Statistical analysis further confirmed that CD45 + and CD3 + A significant decrease in cell number ( Figure 2 I, J), thus highlighting the potential of B16-Exo in modulating immune-mediated transplant rejection.

[0071] Experimental Example 2

[0072] Investigating the inhibition of angiogenesis by B16-Exo

[0073] Neovascularization is a key indicator for evaluating the degree of allogeneic rejection. Immunofluorescence staining for CD31 (an endothelial cell marker) was used to visualize the neovascular network. After image processing of the acquired immunofluorescence micrographs, the perfusion area of ​​the neovascularization was quantitatively evaluated ( Figure 3 A), Image processing for evaluation of neovascularization in a cornea containing a transplant. The cornea containing a transplant is outlined by a dotted circle in the left image (left), capturing the entire corneal area (center), and the right image shows the neovascularized area after treatment; comparative analysis shows that after B16-Exo treatment, the neovascularization level of allogeneic mice returned to baseline levels ( Figure 3 B), thus confirming its inhibitory effect on angiogenesis. In contrast, mice treated with JB6-Exo showed significantly increased corneal neovascularization perfusion area compared with allogeneic controls ( Figure 3 C), indicating that JB6-Exo has limited efficacy in inhibiting neovascularization. This observation shows the potential of B16-Exo as a potent regulator of angiogenesis after corneal transplantation, suggesting its application value in alleviating vascular complications associated with allogeneic rejection.

[0074] To further study the effect of B16-Exo on angiogenesis, we used HUVECs to perform an in vitro tubular structure formation experiment. The results showed that compared with JB6-Exo, B16-Exo significantly inhibited the formation of tubular structures ( Figure 3 D, E), indicating that B16-Exo has a more obvious anti-angiogenic effect.

[0075] Experimental Example 3

[0076] Investigating the regulation of proliferation and function of myeloid-derived suppressor cells by B16-Exo

[0077] First, the biocompatibility of B16-Exo and MDSCs was investigated by CCK-8 assay. After 24 hours of co-culture with B16-Exo, the survival rate of MDSCs was significantly increased ( Figure 7 ), indicating that B16-Exo may promote the survival or proliferation of MDSCs and show good biocompatibility. On day 18 after transplantation, blood and spleen samples were collected from experimental mice for the isolation and subtype characterization of MDSCs ( Figure 8 The results showed that the B16-Exo treatment group had the highest MDSC content in peripheral blood ( Figure 3 F), which was significantly higher than that in the JB6-Exo treated group. This difference was not only manifested in the total MDSC population, but also in the multinuclear (PMN-MDSC) and mononuclear (M-MDSC) subpopulations ( Figure 3 G,H). In addition, a corresponding increase in MDSC levels was also observed in the spleens of B16-Exo-treated mice, exceeding the effect of JB6-Exo treatment ( Figure 3 IK). Therefore, compared with JB6-Exo, B16-Exo has a more significant stimulating effect on MDSC proliferation.

[0078] As a control, we also detected regulatory T cells (Tregs), and there was no significant change in the number of Tregs after treatment ( Fig. 9 ), confirming that MDSCs are the main target cells for exosome-induced immune tolerance. At the same time, the levels of inflammatory mediators in mouse serum were also evaluated to assess the effect of exosomes on MDSC function. Notably, in the B16-Exo-treated group, IL-6, a powerful MDSC stimulator, showed a significant increase ( Figure 3 L). Collectively, these findings reveal the dual abilities of B16-Exo in promoting MDSC expansion and enhancing their function, resulting in a potent immunosuppressive effect.

[0079] Experimental Example 4

[0080] Investigation of the differential expression and potential functional significance of JAK2 in B16-Exo

[0081] Proteomic techniques were used to compare the two exosomes, with a particular focus on differences in protein expression and their potential biological significance. Proteomic sequencing revealed that the two exosomes expressed a total of 2,750 proteins. Notably, B16-Exo uniquely expressed 88 proteins, while JB6-Exo had 29 unique proteins ( Figure 4 A).

[0082] Differential expression analysis showed that 790 proteins were up-regulated and 241 proteins were down-regulated in B16-Exo (fold change ≥ 2, P < 0.05, Figure 4 B). Subsequent protein-protein interaction (PPI) network analysis identified JAK2 as a central upregulated site (degree = 4), emphasizing its key role in the B16-Exo proteome ( Figure 4 C), showing JAK2 as the most frequently interacting protein (degree = 4). Dark blue circles and cyan circles represent up-regulated and down-regulated proteins in the B16-Exo group, respectively (P < 0.05, fold change ≥ 2, top 50 proteins selected). Other proteins without interactions are not shown. The size of the circle is proportional to the number of annotations.

[0083] KEGG pathway analysis mainly associated the gene expression profile of B16-Exo with the JAK-STAT pathway, further reinforcing the core role of JAK2 in the immunomodulatory function of B16-Exo ( Figure 4 D).

[0084] Gene set enrichment analysis (GSEA) was performed to identify signaling pathways significantly associated with the B16 melanoma-derived exosome (B16-Exo) proteome profile. Heat map analysis was performed to facilitate visualization of differentially expressed proteins in the JAK-STAT signaling pathway ( Figure 4E). To verify the proteomic results, we analyzed the expression levels of JAK2 and AKT1 proteins, which are closely related to the JAK-STAT signaling pathway. These two proteins were significantly upregulated in B16-Exo but not in JB6-Exo, which is consistent with our protein heat map data ( Figure 4 F). This further supports the importance of the JAK-STAT pathway in the immunomodulatory effects of B16-Exo.

[0085] GSEA showed significant enrichment of the KEGG JAK-STAT signaling pathway. As shown in the enrichment plot ( Figure 4 G), peak enrichment scores were observed at the top of the ranked indicator score list for B16-Exo, indicating significant overexpression of proteins associated with this pathway in B16-Exo and negative correlation with JB6-Exo. Considering the established association between JAK2 and the enhancement and regulation of MDSC activity, it is plausible that B16-Exo significantly modulates MDSC function through the JAK-STAT signaling pathway. Although KEGG pathway analysis identified the TGF-β signaling pathway as the most enriched pathway, neither B16-Exo nor JB6-Exo expressed TGF-β. However, key components associated with TGF-β signaling, such as SMAD6, ACVR1, ACVRL1, and TGFR1, were enriched in this pathway. Notably, SMAD6 inhibits TGF-β signaling through a feedback mechanism, while ACVR1 and TGFR1 play a key role in mediating TGF-β-induced SMAD1 / 5 phosphorylation. In addition, ACVRL1 is important in endothelial cell function in adult skin vascular remodeling. Considering the key role of MDSCs in immune regulation and anti-angiogenic processes in corneal transplantation, it is conceivable that B16-Exo significantly affects MDSC function through the JAK-STAT signaling pathway, thereby demonstrating its therapeutic effect in controlling immune rejection of allogeneic corneal transplantation.

[0086] Experimental Example 5

[0087] Investigating the expansion and function of MDSCs mediated by B16-Exo through the JAK2 pathway

[0088] MDSCs isolated from mouse tibia were co-cultured with exosomes under the stimulation of IL-6 and GM-CSF, followed by flow cytometry detection and data analysis ( Figure 5 A). Comparative analysis showed that the number of MDSCs increased significantly in the presence of B16-Exo, which is consistent with the findings in animal experiments and further confirms the proliferative effect of B16-Exo on MDSCs. Notably, the addition of anti-JAK2 inhibitors significantly attenuated the activity of B16-Exo ( Figure 5B), resulting in a decrease in the number of MDSCs. Quantitative analysis by flow cytometry also observed a similar trend in PMN-MDSC and M-MDSC subsets ( Figure 5 C,D). These findings collectively demonstrate the prominent role of the JAK2 signaling pathway in mediating the proliferative effect of B16-Exo on MDSCs.

[0089] In addition, to clarify whether B16-Exo regulates T cell proliferation through MDSCs, we co-cultured B16-Exo with MDSCs for three days and then with T cells for five days. The results showed that MDSCs treated with B16-Exo significantly inhibited the proliferation of CD4 + T cell proliferation ( Figure 5 E,F). However, the addition of an anti-JAK2 inhibitor alleviated the effects on CD4 + In contrast, MDSCs pre-treated with JB6-Exo had an inhibitory effect on CD4 + There was no significant effect on the proliferation of T cells, regardless of the presence or absence of anti-JAK2 inhibitors. Therefore, B16-Exo selectively modulates MDSC function through the JAK2 pathway, thereby exerting an inhibitory effect on T cells.

[0090] In summary, B16-Exo strongly enhances the proliferation and immunosuppressive ability of MDSCs through the JAK2 pathway and effectively regulates the proliferation dynamics of T cells.

[0091] Experimental Example 6

[0092] Investigating the role of JAK2 in B16-Exo-induced MDSC expansion and function

[0093] To verify the role of B16-Exo in regulating corneal transplant rejection through the JAK2 pathway in vivo, we performed subconjunctival injection of exosomes followed by continuous injection of JAK2 inhibitors. Compared with the PBS group, B16-Exo significantly improved the transplant survival rate ( Figure 6 A). However, the addition of anti-JAK2 significantly reduced the effect of B16-Exo, confirming the key role of JAK2 in prolonging the survival period after B16-Exo treatment. Observations on corneal transparency and angiogenesis also support these findings. After B16-Exo treatment, corneal transparency was improved and angiogenesis was significantly reduced ( Figure 6 B), Addition of anti-JAK2 partially reversed these parameters, indicating that JAK2 inhibitors may counteract the immunosuppressive effects of B16-Exo.

[0094] In addition, we evaluated the extent of corneal stromal edema. After B16-Exo treatment, the extent of corneal stromal edema in mice was significantly reduced ( Figure 6C). Addition of anti-JAK2 blocked this therapeutic effect, highlighting the importance of JAK2 in the suppression of corneal stromal edema by B16-Exo. Evaluation of immune cell and T cell infiltration showed that mice treated with B16-Exo had increased CD45 in corneal tissue at the graft-host junction and in the center of the graft compared with the PBS group. + Immune cells and CD3 + The infiltration of T cells was significantly reduced ( Figure 6 DF). In contrast, mice in the anti-JAK2 group showed infiltration levels comparable to those in the PBS group. Further vascular fluorescence labeling and quantification of vascular area showed that the addition of anti-JAK2 reversed the reduction in vascular area caused by B16-Exo ( Figure 6 G, H). These results confirm that B16-Exo depends on JAK2 to inhibit corneal transplant rejection in vivo. To explore how B16-Exo affects MDSCs through the JAK-STAT signaling pathway, we analyzed MDSCs and their subtypes in the spleen and blood of mice. After the addition of anti-JAK2, the number of MDSCs and PMN-MDSCs from blood and spleen was significantly inhibited ( Figure 6 IN), supporting the view that B16-Exo depends on JAK2 to promote MDSC function.

[0095] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. For those skilled in the art, improvements and changes obtained without departing from the technical concept of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. Application of melanoma-derived exosomes in the prevention and treatment of corneal transplant immune rejection.

2. The use according to claim 1, characterized in that: The preparation method of melanoma-derived exosomes is as follows: exosomes are isolated from the culture supernatant of a melanoma cell line B16-F10, the cells are cultured in Dulbecco's modified Eagle's medium, supplemented with 10% exosome-removed fetal bovine serum, penicillin and streptomycin, the collected supernatant is centrifuged at 2000xg and 10000xg, filtered, large vesicles, smaller apoptotic bodies and microvesicles are removed in turn, and then ultracentrifuged twice at 100,000xg at 4°C to obtain exosomes.

3. The use according to claim 2, characterized in that: The centrifugation times at 2000×g and 10000×g were 10 min and 60 min respectively.

4. The use according to claim 2, characterized in that: The ultracentrifugation at 100,000 x g was performed twice for 90 min each time.

5. The use according to claim 1, characterized in that: The effective dose of the melanoma-derived exosomes in the drug is 1 μg.

6. The use according to claim 1, characterized in that: The dosage form of the drug includes injection.

Images