CN104726410B - A kind of efflux body and its application with immune suppression function - Google Patents

Abstract

The invention provides an exosome with immunosuppressive function and application thereof. Specifically, the exosomes of the present invention have the following characteristics: (a) the exosomes are vesicle-like structures secreted by immature dendritic cells (imDCs), and the immature dendritic cells dendritic cells are modified with an exogenous membrane-expressed cytokine gene to express said membrane-expressed cytokine; and (b) said exosomes carry said membrane-expressed cytokine. The exosome of the present invention can inhibit the immune response and induce the reconstruction of the body's immune tolerance, thereby preventing and/or treating autoimmune diseases.

Description

Exosome with immunosuppressive function and application thereof

technical field

The invention belongs to the fields of pharmaceutical research and development, cell biology and molecular biology, and relates to the preparation of transmembrane TGF-β1 gene-modified bone marrow immature dendritic cell-derived exosomes and its use in the prevention and treatment of autoimmune diseases Applications.

Background technique

Autoimmune diseases are diseases caused by the body's immune system's immune response to its own components, and have a high incidence in the population. Multiple sclerosis (Multiple Sclerosis, MS) is a common demyelinating disease of the central nervous system that mainly occurs in young people. It is currently believed that it is related to the environment and genetics, but the detailed etiology is unclear. Clinically, the drugs for treating MS mainly include IFN-β, glatiramer, and mitoxantrone, but the effect is not ideal and the side effects are huge.

Dendritic cells (DCs) are professional antigen-presenting cells (Antigen presenting cells, APCs) with the strongest function in the body, which can efficiently ingest, process and present antigens, and immature DCs (immature DCs) have Strong migration ability, mature DCs (mature DCs) can effectively activate naive T cells, and are at the center of initiating, regulating, and maintaining immune responses.

Most DCs in the human body are in an immature state, express low levels of co-stimulatory factors and adhesion factors, and have low ability to stimulate the proliferation of allogenic mixed lymphocytes in vitro, but immature DCs have strong antigen phagocytosis ability. When stimulated by antigens (including in vitro processing) or by certain factors, they can be differentiated into mature DCs, and mature DCs express high levels of co-stimulatory factors and adhesion factors.

During the maturation process, DCs migrate from peripheral tissues exposed to antigens into secondary lymphoid organs, contact T cells and stimulate immune responses. DCs can highly express MHC-I and MHC-II molecules, and MHC molecules bind to the captured and processed tumor antigens to form peptide-MHC molecule complexes and present them to T cells, thereby initiating MHC-I-restricted CTL responses And MHC-class II restricted CD4 ⁺ Thl response. At the same time, DCs also provide the second signal necessary for T cell activation through their highly expressed co-stimulatory molecules (CD80/B7-1, CD86/B7-2, CD40, etc.), and initiate an immune response.

In view of the different roles of mature and immature DCs in the human body, potential drugs for regulating immune responses based on imDCs have been developed in recent years. However, due to the inability to overcome the limitations of MHC-II molecules, these imDCs-based potential drugs all have varying degrees of toxicity to the human body and are less effective.

Therefore, there is an urgent need in this field to develop a safe and effective therapeutic drug that can effectively inhibit autoimmune reactions and regulate immune responses.

Contents of the invention

The present invention provides exosomes from immature dendritic cells with membrane expression type TGF-β1 and not limited by MHC-II, which can induce the reconstruction of immune tolerance of the body, and prevent and treat autoimmune diseases .

The present invention provides a transmembrane type TGF-β1 gene-modified exosomes derived from bone marrow immature dendritic cells, that is, mTGF-β1-EXO, which expresses membrane type TGF-β1. It has a certain immunosuppressive effect in vivo and in vitro and can break through the limitation of MHC-II. It also has a therapeutic effect on experimental autoimmune encephalomyelitis in allogeneic mice, and is expected to become a common preparation for the treatment of autoimmune diseases.

In a first aspect, the present invention provides an exosome, which has the following characteristics:

(a) The exosome is a vesicle-like structure secreted by immature dendritic cells (imDCs), and the immature dendritic cells are modified by exogenous membrane-expressed cytokine genes thereby expressing said membrane-expressed cytokine; and

(b) The exosomes carry the membrane-expressed cytokine.

In another preferred example, the diameter of the exosomes is 50-100 nm.

In another preferred embodiment, the outer surface of the vesicle membrane of the exosome has and displays (at least a part of) the membrane-expressed cytokine.

In another preferred example, the exosome has the activity of inhibiting MHC-II.

In another preferred example, costimulatory molecules CD80, CD86 and MHC-II (major histocompatibility complex II) are underexpressed in the immature dendritic cells.

In another preferred embodiment, the inhibition of the expression of MHC-II is caused or partially caused by the membrane-expressed cytokine.

In another preferred example, the exosomes also have one or more of the following features:

(d) the exosome has immunosuppressive or immune system suppressing activity;

(e) said exosome expressed protein comprises Hsp70, Tsg101, or CD63;

(f) The exosomes do not express the endoplasmic reticulum chaperone Grp94.

In another preferred example, the membrane-expressed cytokines include immunosuppressive cytokines, preferably TGF-β1, IL-10, IL-4, FasL or a combination thereof.

In another preferred embodiment, the exosome has one or more of the following activities:

(i) Inducing the growth of Treg cells (regulatory T cells);

(ii) inhibit Th1 cell polarization;

(iii) Inhibit Th17 cell differentiation.

The second aspect of the present invention provides a pharmaceutical composition, which contains a safe and effective amount of the exosome according to the first aspect of the present invention as an active ingredient; and a pharmaceutically acceptable carrier.

In another preferred example, the pharmaceutical composition is a vaccine composition.

The third aspect of the present invention provides the use of the exosome according to the first aspect of the present invention for preparing a pharmaceutical composition for preventing and/or treating autoimmune diseases.

In another preferred example, the autoimmune disease includes central nervous system demyelinating disease, preferably, the central nervous system demyelinating disease includes multiple sclerosis (Multiple Sclerosis MS), experimental autoimmune disease Encephalomyelitis (experimentalautoimmune encephalomyelitis, EAE), acute disseminated encephalomyelitis, diffuse sclerosis, neuromyelitis optica, and secondary demyelinating diseases.

In another preferred example, the secondary demyelinating disease is mainly caused by systemic diseases, such as periventricular leukomalacia of premature infants and the like.

In the fourth aspect of the present invention, there is provided an immature dendritic cell (Immature Dendritic Cells, imDCs) for producing the efflux body described in the first aspect of the present invention, and the immature dendritic cell is passed through The exogenous membrane-expressed cytokine gene is modified to express the membrane-expressed cytokine, and the immature dendritic cells secrete the exosome according to the first aspect of the present invention, and the Exosomes carry the membrane-expressed cytokines.

In another preferred example, the membrane-expressed cytokines include immunosuppressive cytokines, preferably including TGF-β1, IL-10, IL-4, FasL and the like.

In another preferred example, the expression of MHC-II in the immature dendritic cells is suppressed.

The fifth aspect of the present invention provides a method for preparing exosomes according to the first aspect of the present invention, comprising the steps of:

(a) culturing the immature dendritic cells described in the fourth aspect of the present invention, so as to obtain a mixture of the immature dendritic cells and their secreted efflux bodies;

(b) separating and purifying the secreted exosomes described in (a) from the mixture, so as to obtain the exosomes described in the first aspect of the present invention.

In another preferred example, the separation includes standard four-step centrifugation.

In the sixth aspect of the present invention, an expression vector is provided. The expression vector contains a membrane-expressed TGF-β1 gene, and the membrane-expressed TGF-β1 gene includes TGF-β1 operably linked to a membrane expression guide sequence. Gene.

In another preferred example, the membrane expression guide sequence includes a TM protein sequence and a GPI anchor protein sequence.

In another preferred example, the TM protein sequence includes the TM protein from the HO8910 cell line, whose sequence is shown in SEQ ID NO.: 1; or from the gastric cancer cell line MKN-7, as shown in the gastric cancer cell line The nucleotide sequence Genbank accession number of MKN-7 is X03363.1, GI:31197, CDS is 175-3939, wherein, the nucleotide sequence encoding the transmembrane protein is shown in SEQ ID NO.:9, corresponding to gastric cancer cells The protein of 654-678 of MKN-7, its sequence is shown in SEQ ID NO.:10.

In another preferred example, the expression vector is an adenovirus vector.

In another preferred example, the nucleotide sequence of the transmembrane sequence of the HO8910 cell line is shown in SEQ ID NO.:1; the encoded protein sequence is shown in SEQ ID NO.:4.

In another preferred example, the TGF-β1 gene sequence Genbank accession number NM_011577GI:6755774, its sequence is shown in SEQ ID NO.:2.

The seventh aspect of the present invention provides a method for preventing and/or treating autoimmune diseases, administering a safe and effective amount of the exosomes described in the first aspect of the present invention or the exosomes described in the second aspect of the present invention to the subject in need. said pharmaceutical composition.

In another preferred example, the subject is a mammal, more preferably a human.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows the peak profile of Ad-mTGF-β1 sequencing.

Figure 2 shows the recombinant adenovirus construction strategy.

Figure 3 shows the phenotype of BMDCs after Ad-mTGF-β1 infection, that is, the protein markers on the surface of BMDCs were detected by flow cytometry after adenovirus infection.

Figure 4 shows the function of BMDCs after Ad-mTGF-β1 infection, Figure 4A shows the ability of DCs to resist LPS-induced IL-12P70 secretion after adenovirus infection; Figure 4B shows the ability of DCs after adenovirus infection in mixed lymphocyte reaction role.

Figure 5 shows the phenotype of mTGF-β1-EXO, Figure 5A shows the morphology of exosomes under the electron microscope, scale bar = 200 nm; Figure 5B shows the phenotype of exosomes labeled with fluorescent antibodies and analyzed by flow cytometry; Figure 5C shows the results of WB detection of proteins characteristic of exosomes; Figure 5D shows the expression of TGF-β1 in exosomes detected by flow cytometry; After exosomes, the expression level of CD9 on the latex particles was detected by flow cytometry.

Figure 6 shows that mTGF-β1-EXO treatment can reduce the severity of EAE, and Figure 6A shows the effect of prophylactic administration on the onset of EAE; Figure 6B shows the effect of therapeutic administration on the onset of EAE; Figure 6C shows the effect of EAE onset Mouse brain tissue was stained by H&E to observe the infiltration of inflammatory cells; Lux Fast Blue staining was used to observe the demyelination, the magnification was 400.

Figure 7 shows that mTGF-β1-EXO inhibits MOG-induced T cell proliferation and Th1 polarization, and Figure 7A shows that after MOG peptide stimulates splenocytes or CD4 ⁺ T lymphocytes derived from EAE mice in vitro, cell proliferation is detected by AlamarBlue method . Figure 7B shows the detection of the levels of IFN-γ and IL-10 in supernatants of spleen or CD4 ⁺ T lymphocytes stimulated by MOG peptide by ELISA method.

Figure 8 shows that mTGF-β1-EXO inhibits Th17 differentiation. Figure 8A shows the change of IL-17 in the serum of mice in different treatment groups detected by flow cytometry; Figure 8B shows the change of the proportion of Th17 cells in the spleen of mice in different treatment groups detected by flow cytometry; The changes in the proportion of Th17 cells in the inguinal lymph nodes of mice in different treatment groups were detected.

Figure 9 shows that mTGF-β1-EXO induces Tregs to alleviate the pathogenesis of EAE. Figure 9A shows the proportion of Tregs in spleen and inguinal lymph node lymphocytes of EAE mice after AmTGF-β1-EXO treatment. Figure 9B shows that normal mice were reinfused with CD4 ⁺ CD25 ⁺ Tregs derived from EAE mice in each group and then induced with EAE, and the incidence of EAE in the mice was observed every other day.

Figure 10 shows that mTGF-β1-EXO can attenuate the onset of EAE in allogeneic BALB/c mice. Figure 10A shows that mTGF-β1-EXO derived from C57BL/6 mice inhibits the proliferation of BALB/c mouse T cells in vitro; Figure 10B shows that mTGF-β1-EXO derived from C57BL/6 mice induces BALB/c mice in vitro Mouse Treg; Figure 10C shows the levels of IgE and IgG in the serum of BALB/c mice detected by ELISA 1 week and 4 weeks after reinfusion of exosomes. Figure 10D shows the effect of mTGF-β1-EXO derived from C57BL/6 mice in treating EAE in BALB/c mice.

Detailed ways

After extensive and in-depth research, the inventors prepared for the first time an exosome secreted from immature dendritic cells with immunosuppressive effect. The exosome of the present invention is modified with membrane-expressed immunosuppressive cytokine , there is transmembrane TGF-β1 on the vesicle membrane, which can reflect the nature of imDCs modified by mTGF-β1 gene, and has a strong immunosuppressive function. The mTGF-β1-EXO of the present invention can inhibit the proliferation of CFSE-labeled CD4 ⁺ T lymphocytes and induce the generation of regulatory T cells in large quantities. In addition, it was found that mTGF-β1-EXO could alleviate the symptoms of EAE by down-regulating Th17 cells in the spleen and lymph nodes of mice. Further studies showed that mTGF-β1-EXO inhibited the production of IL-6 by LPS-activated DCs. Sorted CD4 ⁺ Foxp3 ⁺ regulatory T cells in the spleen of mice treated with exosomes in each group of EAE, adopted them into normal mice, and then induced EAE. The results showed that the mTGF-β1-EXO treatment group spleen-derived Treg significantly inhibited the occurrence and development of EAE, while LacZ-EXO derived from DCs transfected with Ad LacZ, Control-EXO derived from DCs in the untreated group, sTGF-β1-EXO, and Treg derived from the untreated group significantly inhibited inflammation. weaken. More importantly, mTGF-β1-EXO derived from C57BL/6 mice can break through the limitation of MHC, and can also have a therapeutic effect on allogeneic PLP-induced EAE in BALB/c mice, so it is expected to be a therapeutic agent. A universal agent for autoimmune diseases. On this basis, the present invention has been accomplished.

In addition, the present invention also provides a pharmaceutical composition containing the exosome of the present invention and its therapeutic use.

Exosomes

Exosomes are small vesicles secreted by various living cells, non-plasma membrane-derived, with a diameter of 50-100 nm and a lipid bilayer structure. In addition to the common markers of exosomes from different cells, such as CD9, CD63, CD81 and CD82, heat shock protein HSP70, HSP90, Tsg101, Alix and other protein molecules, this small vesicle also contains MHC-I, which reflects the characteristics of imDCs cells. MHC-II molecules, CD80, CD86, ICAM-I and other special proteins, and the expression is lower than that of mDCs.

membrane expressed cytokine

Most of the cytokines in the body are secreted, but a few cytokines exist on the cell surface in the form of transmembrane. Studies have shown that the biological activity and mode of action of secreted and transmembrane cytokines are not consistent. For example, transmembrane TNF-α acts locally through cell-to-cell contact, while secreted TNF-α can enter the blood circulation and act on distant tissues and organs, and transmembrane TNF-α is more active than secreted TNF-α. The tumor killing spectrum is wider, and it can effectively kill tumor cells resistant to secreted TNF-α. In addition, the ability of secreted TNF-α and membrane TNF-α expressed on CD4 ⁺ T cells to activate macrophage defense against Leishmania was also significantly different.

The membrane-expressed cytokines that can be used in the present invention are membrane-expressed cytokines with immunosuppressive function, and the membrane-expressed cytokines can be naturally occurring or artificially expressed. A preferred artificially synthesized and expressed membrane-expressed cytokine can be formed by splicing an immunosuppressive cellular gene with the coding sequence of the transmembrane region of a specific cell.

A preferred immunosuppressive cytokine useful in the present invention is TGF-β1. In view of the powerful immune regulation function of TGF-β1 and the characteristics of exosomes derived from imDCs, secreted cytokines are likely to be encapsulated by exosomes, which will lead to the impairment of TGF-β1 function.

The TGF-β1 adenovirus vector (Adenovirus expressing membrane-associated TGF-β1, Ad mTGF-β1) containing TM transmembrane segment constructed by molecular bridging method in the present invention can be seen from the amino acid properties of the encoded TM region. Most amino acids are hydrophobic, which facilitates their transmembrane interaction. Exosomes derived from Ad mTGF-β1 gene-modified imDCs were extracted and found to reflect the properties of mTGF-β1 gene-modified imDCs, have a strong immunosuppressive function, and further alleviate the symptoms of EAE.

In addition, linking other immunosuppressive cytokines to transmembrane proteins can also be used to generate exosomes derived from imDCs containing membrane-expressing forms. For example, immunosuppressive cytokines useful in the present invention also include TGF-β1, IL-10, IL-4, FasL or combinations thereof. The TM protein sequence may also include the TM protein from the HO8910 cell line, whose sequence is shown in SEQ ID NO.: 1; or the TM protein from the gastric cancer cell line MKN-7, whose sequence is shown in SEQ ID NO.: 10, the nucleotide sequence encoding the protein is shown in SEQ ID NO.:9.

TGF-β1 and membrane-expressed TGF-β1 (mTGF-β1)

Transforming Growth Factor-beta1 (TGF-beta1) is a dimeric protein containing 112 amino acids and a molecular weight of 25KD (Genbank accession number AAH13738GI: 15489275, its protein sequence is shown in SEQ ID NO.: 3, encoding the protein The nucleotide sequence is shown in SEQ ID NO.:2.

TGF-β1 (highly conserved among various genera, for example, humans, rats, and mice have exactly the same amino acid sequence. Almost all cells have receptors for TGF-β1, and its influence on cell growth and differentiation is very extensive.

TGF-β1 plays an important role in maintaining immune homeostasis. TGF-β1-deficient mice have severe multi-organ inflammatory responses and abnormally elevated MHC in multiple tissues. Blocking TGF-β1 signaling in T cells, mice suffered from an autoimmune disease characterized by inflammatory cell infiltration in the lungs and colon, and autoimmune antibodies were detected in the blood.

As used herein, the terms "membrane-expressed TGF-β1", "transmembrane TGF-β1" and "membrane-bound TGF-β1" are used interchangeably and refer to the N-terminus of imDCs connected to the cell's transmembrane segment TM Located outside the cell membrane, the C-terminal is located inside the cell membrane and can directly act on the TGF-β1 factor of the cell. A preferred nucleotide sequence of membrane-expressed TGF-β1 is shown in SEQ ID NO.:11.

In the present invention, the membrane-expressed TGF-β1 can be located in the cells or on the cell membrane of imDC cells. Preferably, about 20-30% of TGF-β1 can be localized and expressed on imDCs, and its N-terminus is located outside the cell membrane. Exosomes are lipid bilayer vesicles with a diameter of 50-100 nm released by living cells. After they combine with a subcellular organelle multivesicular body (MVBs) in the cell, they form exosomes through inner membrane budding , and then exosomes are released into the extracellular matrix through the direct fusion of MVBs with the cell membrane. Therefore, Exosomes carry some proteins and molecules in the cell and on the cell membrane during the process of secreting from the cytoplasm to the outside. Exosomes have multiple functions depending on the nature of the cells from which they originate.

Experiments show that mTGF-β1-EXO reduces antigen-specific Th1 response and IFN-γ production, but promotes IL-10 production; mTGF-β1-EXO reduces Th17 production in EAE mice, which may be related to the downregulation of P38 in activated DCs , Erk, Stat3 and NF-κB expressions, which lead to the decrease of IL-6; in addition, mTGF-β1-EXO increases the production of Treg in spleen and lymph nodes, and the CD4 ⁺ Foxp3 ⁺ regulatory T cells in the adoptive transfer mTGF-β1-EXO treatment group are significantly reduced Onset of EAE in mice.

immature dendritic cells

Dendritic cells (Dendritic Cells, DCs), as the most powerful professional antigen-presenting cells, play a role in the occurrence and progression of EAE.

Mature DCs express high levels of co-stimulatory molecules and major histocompatibility complex MHC-II, induce the differentiation and activation of Th1, Th17, B cells and macrophages and other pro-inflammatory cells, and the activated autoreactive inflammatory cells and The cytokines and demyelinating antibodies produced by it enter the CNS from the systemic circulation through the damaged blood-brain barrier. These cells trigger a series of events by secreting pro-inflammatory cytokines, resulting in immune response-mediated damage to myelin and microglia, so that axons are exposed and cannot effectively conduct action potentials, resulting in neurological symptoms.

Immature DCs (immature Dendritic Cells, imDCs) express low-molecular-weight MHC-II and co-stimulatory molecules, which can induce T cell dysfunction in vitro, and can also induce CD4 ⁺ CD25 ⁺ regulatory T (regulatory T cells, Tregs) cells, Th3 Or Tr1 differentiation, these cells relieve the symptoms of EAE by secreting cytokines such as IL-10, TGF-β1, soluble HLA-G, or exerting immune regulation function by contacting with T cells or interacting with DCs. However, imDCs may further mature under the action of inflammatory environment, thus forming mDCs.

autoimmune disease

Autoimmune diseases refer to diseases caused by the body's immune response to self-antigens, resulting in damage to its own tissues. In the present invention, autoimmune diseases include central nervous system demyelinating diseases or local inflammatory diseases.

As used herein, demyelination of the central nervous system refers to a group of acquired disorders with different etiologies and clinical manifestations, but with similar characteristics, characterized by pathological changes in which nerve fibers are demyelinated and nerve cells are demyelinated. relatively intact. Because the common cause of central nervous demyelinating lesion is central nervous local inflammatory lesion, or central nervous demyelinating lesion can coexist with local inflammatory lesion, therefore, central nervous demyelinating lesion of the present invention also includes the demyelinating lesion that local inflammatory lesion causes. Myelin disorders.

In another preferred example, the central nervous system demyelinating diseases include multiple sclerosis (MultipleSclerosis, MS), experimental autoimmune encephalomyelitis (Experimental AutoimmuneEncephalomyelitis, EAE), acute disseminated encephalomyelitis, diffuse Cerebral sclerosis, neuromyelitis optica, etc.; secondary demyelinating diseases are mainly caused by systemic diseases, such as periventricular leukomalacia in premature infants. In experimental zoology, the most widely used and typical animal model of central nervous system demyelinating disease is experimental autoimmune encephalomyelitis.

experimental animal model

Experimental Autoimmune Encephalomyelitis (EAE) is an autoimmune disease characterized by localized inflammation and demyelination of the central nervous system (CNS), and is a study of multiple sclerosis (multiple sclerosis). , MS) an important experimental model of pathogenesis and treatment options.

Studies have shown that dendritic cell TGF-β receptor signal inactivated mice were immunized with MOG _35-55 , and a large number of T cells infiltrated the nervous system, and there were high levels of Th1 and Th17 cytokines in the periphery, and the symptoms of EAE could not be relieved. . Crossbreeding with autoimmune-prone MOG-TCR mice showed spontaneous EAE symptoms characterized by early activation of a large number of myelin-specific T cells, microglial infiltration, mouse movement disorders, and premature death .

A certain dose of TGF-β1 administered by nasal feeding can inhibit delayed-relapse EAE. Further studies have shown that this is achieved by TGF-β1 inhibiting the production of nitric oxide by DCs, which in turn can induce T cell apoptosis. TGF-β1 can also promote the production of IL-10+IFNγ+ Th1 cells with immunomodulatory effects through Smad4, which can prevent the recruitment of inflammatory cells such as Th1 and Th17 to the central nervous system by secreting IL-10, thereby alleviating the symptoms of EAE .

pharmaceutical composition

The invention also provides a pharmaceutical composition. The pharmaceutical composition contains a pharmaceutically acceptable carrier (including diluents, excipients, etc.) and an effective amount of the exosome of the present invention as an active ingredient. The amount of exosomes of the present invention is usually 10 μg-100 mg/dose, preferably 100-1000 μg/dose.

As used herein, the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or exhibits a detectable therapeutic or preventive effect. The precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the symptoms, and the therapeutic agent(s) and/or combination of therapeutic agents chosen for administration.

For the purposes of the present invention, an effective dose is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg body weight of exosomes of the present invention administered to an individual.

The pharmaceutical composition also contains a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent (eg, exosomes). The term refers to pharmaceutical carriers which do not, by themselves, induce the production of antibodies deleterious to the individual receiving the composition and which are not unduly toxic upon administration. These vectors are well known to those skilled in the art.

Pharmaceutically acceptable carriers in therapeutic pharmaceutical compositions can contain liquids such as water, saline, glycerol and ethanol. In addition, there may also be auxiliary substances in these carriers, such as wetting agents or emulsifying agents, pH buffering substances and the like. In addition, immune adjuvants may also be included in the immune composition.

Typically, therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution, or suspension, in liquid carriers prior to injection can also be prepared.

Once formulated, the compositions of the invention can be administered directly to a subject. The subject to be prevented or treated may be an animal, especially a human.

The therapeutic or preventive pharmaceutical composition containing exosomes of the present invention can be administered orally, subcutaneously, intradermally, intracavitally, at the lesion site, intra-articularly, or intravenously. The therapeutic dosage regimen can be a single-dose regimen or It can be a multi-dose regimen.

Beneficial effects of the present invention:

1. The exosome of the present invention has a good immunosuppressive effect, thereby treating autoimmune diseases.

2. The exosome vesicle membrane of the present invention has or displays membrane-expressed TGF-β1, which can effectively inhibit the proliferation and differentiation of immune cells, induce Treg cell differentiation, and treat EAE.

3. The exosome of the present invention can inhibit the expression of MHC-II, so that it is not restricted by MHC-II, fully exerts the effect of suppressing immunity, and is safe and effective.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Example 1 Construction of recombinant replication-deficient adenoviral vector containing membrane-expressed TGF-β1 gene, and packaging of recombinant adenovirus Ad-mTGF-β1

method:

By means of PCR amplification, using the cDNA of 3LL cells (given by the Institute of Immunology, Shanghai Second Military Medical University) as a template, primers were designed according to the full-length gene sequence of human TGF-β1, from ovarian cancer cell line HO8910 cells (given by Shanghai Second Military Medical University). (Genbank accession number: M11730.1; GI: 183986) encoding transmembrane (Transmembrane, TM) The specific primers (as shown in SEQ ID NO.: 7 and 8) of the region fragment (2041-2161), PCR amplified the TM fragment; the splicing overlap extension (SplicingOverlap Extension, SOE) method was used to connect TGF-β1 and TM two gene fragments; the fusion gene TM-TGF-β1 is directionally connected with the cloning vector pDC315.

After the sequencing is correct, the target gene mTGF-β1 recombinant shuttle plasmid and the plasmid with the virus backbone are co-transfected into the packaging cell Hek293, and the recombinant adenovirus is obtained by homologous recombination (see Figure 1). details as follows:

1) Extract total RNA from 3LL cells and use TGF-β1 primer

Forward: 5'-ATTGAATTCATGCCGCCCTCGGGGCTGCGGCTAC-3' (SEQ ID NO.:5);

Reverse: 5'-GCTCTCTGCTCGGCGCTGCACTTGCAGGAGCGCA-3' (SEQ ID NO.: 6) for reverse transcription;

Extract total RNA from HO8910 cells and perform reverse transcription with TM primers

Forward: 5'-TGCGCTCCTGCAAGTGCAGCGCCGAGCAGAGAGC-3' (SEQ ID NO.:7);

Reverse: 5'-CGTGTCGACCGTGTACTTCCGGATCTTCTGCTGC-3' (SEQ ID NO.: 8).

TGF-β1 fragment PCR reaction conditions were: 95°C for 5min; 94°C for 30s, 60°C for 30s, 72°C for 80s, 35 cycles of amplification; 72°C for 10min.

The TM fragment PCR reaction conditions were: 95°C for 5 min; 94°C for 30 s, 63°C for 30 s, 72°C for 11 s, 35 cycles of amplification; 72°C for 10 min. After the completion of PCR, the corresponding bands were cut out, and the gel was recovered.

2) Measure the concentration of TGF-β1 and TM recovered from the gel, add the two in an equimolar ratio, use the front chain of TGF-β1 and the back chain of the TM segment as primers, and perform bridging PCR to construct the fusion gene mTGF-β1 .

The PCR reaction conditions were 95°C for 5min; 80°C for 3min; 94°C for 30s, 55°C for 40s, 72°C for 90s, 35 cycles of amplification; 72°C for 10min.

3) The target band was recovered from the gel, and pDC315 plasmid and TGF-β1-Tm were digested with endonucleases ECOR I and Sal I, connected at 16°C overnight, and 6 colonies were randomly selected for PCR identification after YC transformation. Colony Shake. After 16 hours, the plasmids of the positive bacteria identified by PCR were extracted and identified by enzyme digestion, and the positive ones were sent to the company for two-way sequencing. The results are shown in Figure 2.

4) Hek293 was plated at 4×105 cells/well 24 hours before transfection. After the cell confluence was greater than 80%, 1 ml of serum-free DMEM medium was replaced. At the same time, 10 microliters of liposomes, 3 μg pBHGloxΔE1, 3 μg Cre (provided by Shanghai Donated by the Institute of Immunology, Second Military Medical University), 2 μg of pDC315 containing the TGF-β1-Tm gene fragment, and 1 ml of DMEM medium was supplemented 6 hours later. Observe cell changes and replenish fluid in time. After 10 days, the cells and supernatant were collected for PCR identification and flow cytometric identification.

Example 2: Induction, amplification and adenovirus infection of BMDCs

BMDCs were cultured, and after being infected with Ad-mTGF-β1 for 24 hours, the residual adenovirus was washed with PBS, BMDCs were cultured for 48 hours, and the supernatant was collected for extraction of mTGF-β1-EXO. The specific operation is as follows:

1) Culture of BMDCs: Dendritic cells were derived from normal six-week-old female C57BL/6(H-2Kb) mice. The femur and tibia of the mice were aseptically removed, and the bone marrow cells were washed out with serum-free RPMI1640, and treated with Tris-NH4Cl After destroying red blood cells, culture them in RPMI1640 containing 10% fetal bovine serum, add 20ng/ml GM-CSF, 1ng/ml IL-4 and culture them at 37°C, 5% CO ₂ for 48 hours, then use pre-warmed culture The non-adherent cells were washed away, and the adherent cells were cultured with DC medium for 48 hours, and the DCs harvested on the fifth day were immature DCs by gently pipetting.

2) Infection of BMDCs with Ad-mTGF-β1: count DCs accurately on the fifth day of culture, and then use Ad-sTGF-β1 (secreted TGF-β1) and Ad-mTGF-β1 (transmembrane TGF-β1) or Ad- Cells were transfected with Lac Z at MOI (multiplicity of infection, MOI)=100, and a blank control group without virus transfection was set up.

The transfection process was as follows: 1×10 ⁷ /ml DCs were resuspended in a 2ml eppendorf tube with serum-free medium, the corresponding virus solution was added, and shaken at 60 rpm for 2 hours on a constant temperature shaker at 37°C. After 2 hours, the complete medium was supplemented with 2×10 ⁶ /ml, placed in a 37°C incubator for 18 hours, and then the medium was removed by centrifugation, and washed twice with PBS to wash away residual virus. Afterwards, the cells were further cultured in the DC medium, and the fetal bovine serum contained in the medium was ultracentrifuged at 100,000g×1h to remove serum-derived exosomes. After 48 hours, the cells were collected for phenotype and function analysis, and the cell culture supernatant was saved for the detection of cytokines and the preparation of exosomes.

Example 3: Isolation and purification of mTGF-β1-EXO

The previously retained cell culture supernatant was prepared by a standard four-step centrifugation method to prepare exosomes. details as follows:

Centrifuge at 300g×10min, 1200g×20min, 10000g×20min at high speed at 4°C to remove cell debris and other aggregated substances; then ultracentrifuge at 100000g×60min, resuspend the obtained pellet in a large amount of PBS and ultracentrifuge again at 100000g×60min to remove residual culture medium, the resulting precipitate is relatively pure exosomes. The exosomes were resuspended in a small amount of PBS, and the protein content was determined by the BCA method. The isolated exosomes were aliquoted and stored at -80°C.

Identification of phenotypes after embodiment 4 BMDCs infection with Ad-mTGF-β1

Flow cytometry was used to detect the expression of surface molecules of DCs transfected with different adenoviruses and continued to culture for 24 hours. The operation was as follows: DCs of different treatment groups were collected, 1×10 ⁶ DCs were resuspended in 100 μl PBS, Add corresponding doses of fluorescently labeled antibodies: MHC-II, CD80, CD86, CD40, and CD11c according to the instructions, incubate on ice for 30 minutes, wash with PBS three times, detect with flow cytometry, and analyze with CellQuest software.

The results are shown in Figure 3: wherein, Ad-sTGF-β1 and Ad-LacZ represent the control group infected with adenovirus containing secreted TGF-β1 and LacZ sequences respectively, and Control is the group without adenovirus infection.

The results showed that all groups expressed mouse DCs surface marker molecule CD11c, and the expression of MHC-Ⅱ molecules and co-stimulatory molecules such as CD86 and CD80 on the surface of DCs in the control group that were not transfected with adenovirus were very low, which was in line with the characteristics of immature DCs. Phenotypic characteristics. Infection with Ad LacZ did not change the phenotypic characteristics of the cells, suggesting that Ad-LacZ does not affect the maturation of DCs. After Ad-mTGF-β1 transfection, the expressions of MHC-II and CD80 on the surface of DCs were all inhibited, and the inhibitory effect on MHC-II was the most obvious. Although Ad sTGF-β1 could also inhibit the expression of MHC-II on DCs, less effective than the former.

Identification of the function of BMDCs infected with Ad-mTGF-β1 in embodiment 5

The expression levels of MHC molecules and co-stimulatory molecules such as CD86 on the surface of immature DCs are very low, which cannot provide the necessary activation signals for T cells, so their ability to activate T cells and induce immune responses is very weak. Immature DCs can rapidly mature and secrete IL-12 under the stimulation of LPS. Therefore, in order to observe the changes in the function of BMDCs infected with Ad-mTGF-β1, this embodiment detected the changes in the secretion of IL-12p70 of BMDCs transfected with different adenoviruses under LPS stimulation and its role in the mixed lymphocyte reaction, specifically as follows:

1) ELISA detection of IL-12p70: DCs transfected with adenovirus for 24 hours and DCs not transfected with adenovirus were collected from the above groups and stimulated with 1 μg/ml lipopolysaccharide (LPS), and a corresponding non-stimulation control was set up groups, with 3 replicate wells in each group. The culture was continued for 24 hours, the culture supernatant was collected, and the concentration of IL-12p70 was detected by double-antibody sandwich ELISA.

2) Mixed lymphocyte reaction: Take the spleen of BALB/c(H-2Kd) mice, prepare the spleen cell suspension of the mice aseptically, sort out CD4 ⁺ T cells with magnetic beads, and adjust the concentration of CD4 ⁺ T cells to 2× 10 ⁶ /ml, added to a 96-well plate at a volume of 100 μl per well. Take the DCs from each group derived from the bone marrow of C57BL/6(H-2Kb) mice 24 hours after transfection with the adenovirus, add 50 μg/ml mitomycin C, inactivate in a water bath at 37°C for 30 minutes, wash twice with PBS, Adjust its concentration to 2×10 ⁵ /ml, add DC:T=1:10, 1:20, 1:40 to a 96-well plate, make up the medium to make the final volume 200 μl, and set up triplicate wells for each group. After incubation at 37°C for 4 days, 20 μl of alamar Blue dye was added to each well for staining. After continuing to incubate for 24 hours, the fluorescence intensity was detected with an excitation wavelength of 535nm and an emission wavelength of 595nm.

The result is shown in Figure 4:

Figure 4A shows that the level of IL-12p70 secreted by DCs transfected with Ad-mTGF-β1 and Ad-sTGF-β1 is lower than that of the control group. After stimulation with LPS, untreated Control DCs and LacZ DCs can mature rapidly and secrete IL The level of -12p70 was significantly increased. However, the IL-12p70 secretion level of Ad-mTGF-β1-DCs and Ad-sTGF-β1-DCs increased slightly, indicating that DCs transfected with Ad-mTGF-β1 and Ad s-TGF-β1 induced maturation of LPS, etc. Factors have resistance, but the former is stronger than the latter.

Figure 4B shows that after transfection with Ad-mTGF-β1 and Ad-sTGF-β1, the ability of DCs to stimulate T cell proliferation was significantly inhibited. Therefore, BMDCs transfected with Ad-mTGF-β1 have the functional characteristics of immature DCs.

Phenotype identification of embodiment 6mTGF-β1-EXO

mTGF-β1-EXO was isolated, and its phenotype was analyzed by transmission electron microscopy, Western Blot, and flow cytometry, as follows:

6.1 Electron microscope analysis of exosomes: Wash the exosome suspension twice with PBS, fix with fixative solution (2% paraformaldehyde, 0.25% glutaraldehyde) at 4°C for 1 hour, wash once with PBS to make suspension drops The slices were fixed with 1% osmic acid, dehydrated with graded alcohol, and the ultrathin sections were stained with lead and uranium, observed and photographed under a transmission electron microscope.

The results are shown in Figure 5A, the exosomes we isolated had a typical lipid bilayer structure, and were all small vesicles with diameters concentrated in the range of 50-100 nm.

6.2Western blot analysis: the protein concentration of exosomes was determined by BCA method, adding loading buffer, boiled in boiling water for 5min, and separated by 12% SDS-PAGE electrophoresis, adding 20 μg of exosomes to each well, and electrotransferred to nitrocellulose On the membrane, after blocking with 5% skimmed milk, add mouse anti-mouse Hsp70, anti-mouse Grp94, goat anti-mouse Tsg101, rabbit anti-mouse TGF-β1 polyclonal antibodies respectively, combine at 4°C overnight, PBST After fully washing, add the corresponding secondary antibody to bind for 1.5 hours, and develop according to the instructions of the enhanced chemiluminescence kit.

The results are shown in Figure 5-C, where Lac-Z-EXO and sTGF-β1-EXO refer to the separation and purification of exosomes from the supernatant of BMDCs infected with Ad-LacZ and Ad-sTGF-β1, respectively.

The results showed that exosomes in each group expressed exosome-associated proteins Hsp70, Tsg101, and CD63, but did not express endoplasmic reticulum chaperone Grp94, and only mTGF-β1-EXO and sTGF-β1-EXO expressed TGF-β1.

6.3 FACS identification of exosome phenotype: Add 5×10 ⁵ /100 μl latex particles in a saturated amount of exosomes, incubate at room temperature for 30 min, add PBS to a final volume of 1 ml, shake at 37°C overnight. Then, the latex particles adsorbing exosomes were evenly divided, and fluorescently labeled antibodies: TGF-β1, MHC-II, CD80, CD86, CD40, and CD11c were added respectively, with a final concentration of 5 μg/ml, incubated at room temperature for 20 min, and washed twice with PBS. After the first pass, the cells were detected by flow cytometry and analyzed by CellQuest software.

The results are shown in Figure 5B: CD11c, a characteristic marker molecule on the surface of mouse DCs, was expressed in the exosomes of each group, MHC-II was almost not expressed in mTGF-β1-EXO, and CD80 was also down-regulated compared with the control group. This also shows that exosomes can reflect the characteristics of the DCs from which they originate, and have certain immunoregulatory effects.

6.4 Validation of the expression pattern of TGF-β1 on exosomes: TGF-β1 expression was detected on mTGF-β1-EXO but not sTGF-β1-EXO (Fig. 5D). In order to further verify the expression form of TGF-β1 on exosomes, 5 μl latex particles were incubated with 50 μg anti-TGF-β1 monoclonal antibody at room temperature for 1 h, then centrifuged, blocked with FCS, anti-TGF-β1 mAb Coated latex particles were shaken overnight at 4°C with 20 μg of exosomes. After washing with PBS, PE-anti-CD9 antibody was labeled, and the expression of CD9 was detected by FACS.

The results are shown in Figure 5E: mTGF-β1-EXO can be captured by latex particles coated with anti-TGF-β1 mAb, and CD9 expression is positive in FACS detection, while sTGF-β1-EXO expressing secreted TGF-β1 does not express CD9, This further shows that TGF-β1 in mTGF-β1-EXO is membrane expressed.

Example 7 Evaluation of mTGF-β1-EXO prevention and treatment can reduce the effect of EAE

The mouse model of EAE was used, and mTGF-β1-EXO was used to prevent and treat it, and its effect was evaluated. details as follows:

1) EAE induction and assessment of disease progression

Mix MOG35-55 (purchased from Shanghai Sangong) or PLP _180-199 (purchased from Shanghai Sangong) with tuberculin and an equal volume of complete Freund's adjuvant, and beat repeatedly to form a stable water-in-oil antigen emulsion. Each C57BL/6 mouse was subcutaneously injected with 200 μg of MOG _35-55 and 200 μg of tuberculin at three points, and each Balb/c mouse was subcutaneously injected with 300 μg of PLP _180-199 and 200 μg of tuberculin at three points, and after immunization At 0 and 48 hours, 200 ng of botulinum toxin was injected into the tail vein.

The mice were evaluated by a double-blind method, observed continuously for 36 days, and scored according to the following criteria for neurological function: 0 points, no abnormality; 1 point, decreased tail tension; 2 points, hindlimb paresis, gait instability and / or swinging; 3 points, incomplete hindlimb paralysis; 4 points, complete hindlimb paralysis; 5 points, near-death state, hindlimb paralysis accompanied by forelimbs. We found that the mice started to get sick on the 10th day, and the EAE mice got sick on the 14th-16th day. In severe cases, the clinical manifestations were paralysis of the hind limbs, and even death.

2) Prevention and treatment of EAE mice

The 6-8 week old female C57BL/6 were equally divided into five groups, 10 in each group, grouped as follows:

mTGF-β1-EXO

sTGF-β1-EXO

LacZ-EXO

Control-EXO

no treatment

8 days, 5 days and 2 days before modeling, exosomes of each group were injected via tail vein at 10 μg/rat, EAE was induced on day 0, scores were recorded, and mTGF-β1-EXO and sTGF-EXO were observed and compared. Preventive effect of β1-EXO on EAE. Further test the therapeutic effects of mTGF-β1-EXO and sTGF-β1-EXO on EAE, inject the exosomes of each group with the same dosage as the prevention group on the 14th day, 17th day and 21st day of the peak period of onset, and observe the score .

The results are shown in Figures 6A and 6B: the preventive intervention of mTGF-β1-EXO and sTGF-β1-EXO can significantly reduce the score of EAE, but the effect of the former is more obvious than that of the latter (Figure 6A). After EAE was modeled, mTGF-β1-EXO treatment on days 14, 17 and 21 could significantly improve the symptoms of EAE, and the clinical score was lower than that of other treatment groups, and some mice even completely relieved (Fig. 6-B).

3) Histopathological detection of EAE mice

After two weeks of treatment, the heart was perfused with 4% paraformaldehyde, the brain tissue was quickly separated, fixed in 4% neutral formaldehyde fixative solution, embedded in paraffin and made into 6 micron thick sections, routine hematoxylin-eosin (HE) staining and tar Violet staining (Luxol fastblue, LFB), and observed inflammatory cell infiltration and demyelination changes under a light microscope.

The results are shown in Figure 6C: mTGF-β1-EXO significantly reduced the inflammatory cell infiltration and demyelination in the lateral ventricles of EAE mice, while the sTGF-β1-EXO treatment group had no such effect.

Conclusion: The above results show that mTGF-β1-EXO has a strong immunosuppressive ability and can effectively prevent and inhibit the occurrence and development of EAE in C57BL/6 mice.

Example 8 Evaluation of the effect of mTGF-β1-EXO on inhibiting MOG-induced T cell proliferation and Th1 polarization

Autoreactive T cells and Th1 cells are closely related to the progression of EAE, so this example observes the effect of mTGF-β1-EXO on T cell proliferation and Th1 polarization induced by MOG.

method:

After two weeks of Exosomes treatment, the spleen cells and CD4 ⁺ T cells of mice in each treatment group were separated, 2×10 ⁵ /well were spread on a 96-well plate, and 10 μg/ml MOG _35-55 was added. In the CD4 ⁺ T cell group, Add 2×10 ⁴ mitomycin C-inactivated DCs to each well at a ratio of 1:10, set up three replicate wells in each group, add 20 μl alamarBlue, DTX880 multimode detector (Beckman Coulter, PaloAlto, CA) after 48 hours of culture Check its fluorescence intensity. After culturing for 72 hours under the same culture conditions, the supernatants of each group were collected, and the expression levels of IL-10, IL-6, IL-17 and IFN-γ in the supernatants of each group were detected by ELISA.

The results showed that after being stimulated with 10 μg/ml MOG peptide for 3 days in vitro, the proliferation of spleen and CD4 ⁺ T lymphocytes derived from EAE mice treated with mTGF-β1-EXO could be observed, which was significantly higher than that of mice in other treatment groups. was inhibited (Fig. 7A). In addition, mTGF-β1-EXO treatment can also inhibit the secretion of IFN-γ in the spleen and CD4 ⁺ T lymphocytes derived from EAE mice stimulated by MOG peptide in vitro, and promote the secretion of IL-10 (Fig. 7B).

Conclusion: The therapeutic effect of mTGF-β1-EXO on EAE may be related to the inhibition of proliferation and Th1 polarization of autoreactive Th cells.

Example 9 Evaluation of mTGF-β1-EXO Inhibiting Th17 Differentiation Effect

After two weeks of treatment, the serum of the mice was taken to detect the level of IL-17 by ELSA, and the lymphocytes in the spleen and inguinal lymph nodes of the mice were isolated to detect the distribution of Th17.

Results As shown in Figure 8, the expression of IL-17 in mTGF-β1-EXO-treated mice was significantly lower than that of other groups, and the ratio of Th17 cells in spleen and lymph nodes was also significantly lower than that of other groups. This indicates that the therapeutic effect of mTGF-β1-EXO on EAE may be related to its inhibition of Th17 cells.

Example 10 Evaluation of the effect of mTGF-β1-EXO inducing Treg to reduce the incidence of EAE

This example examines whether mTGF-β1-EXO can induce the generation of Tregs in EAE mice.

10.1

Methods: Tregs are important regulators of autoimmune diseases. First, we tested whether mTGF-β1-EXO has the function of inducing Tregs in EAE mice. After two weeks of exosomes treatment, we isolated the spleen and lymph nodes of mice in each treatment group, and labeled anti-CD4 and anti-Foxp3 flow antibodies. The ratio of CD4 ⁺ Foxp3 ⁺ regulatory T cells was detected by FACS.

The results are shown in Figure 9A: mTGF-β1-EXO treatment can significantly increase the proportion of Tregs in spleen and inguinal lymph node lymphocytes of EAE mice.

10.2 In order to further clarify the protective effect of mTGF-β1-EXO-induced Tregs on EAE mice, Foxp3-GFP reporter mice were used as the EAE model. After exosomes were treated for two weeks, the spleen and lymph nodes of each treatment group were sorted by flow cytometry. The purity of CD4 ⁺ Foxp3 ⁺ Treg cells was 89%-92%. Adoptive transfer into normal mice by tail vein injection at a dose of 1×10 ⁶ /mouse, induced EAE two days later, and observed and recorded scores .

The results are shown in Figure 9B: CD4 ⁺ Foxp3 ⁺ Tregs from normal mice and mTGF-β1-EXO-treated EAE mice could significantly reduce the onset of newly induced EAE mice.

The results showed that mTGF-β1-EXO-induced Tregs had a protective effect on EAE mice.

Example 11 Evaluation of the effect of mTGF-β1-EXO alleviating the onset of EAE in allogeneic BALB/c mice

11.1 First, detect whether mTGF-β1-EXO derived from C57BL/6 mice can inhibit the proliferation of BALB/c mouse T cells and induce Tregs in vitro.

Method: CD4 ⁺ T cells in the spleen of BALB/c mice were sorted by magnetic beads, 1×10 ⁷ CD4 ⁺ T cells were resuspended in 100 μl of PBS, and CFSE (5,6-carboxyfluorescein diacetate, 5,6-carboxyfluorescein diacetate, succinimidylester, CFSE), after incubating at 37°C for 3 min, immediately wash twice with pre-cooled 1640 complete medium containing 10% calf serum, adjust the concentration to 1×10 ⁶ /ml, add 1 μl anti-CD3/ CD28 magnetic beads, 200μl/well spread in 96-well plate, then add 0.5μg/ml, 1μg/ml, 2μg/ml exosomes of different groups, and set up a separate group without any exosomes as a control, each group set Three replicate wells were incubated at 37°C for 3 days, and then the fluorescence intensity was detected by FACS.

In the experiment of Treg differentiation induced by Exosomes, first magnetic beads were used to sort BALB/c mouse splenic CD4 ⁺ T cells, 1 μl anti-CD3/CD28 magnetic beads were added to 1×10 ⁶ /ml CD4 ⁺ T cells, and 200 μl was added to 96 orifice plate. At the same time, exosomes of each group acidified by 1N HCL were added or not added at 2 μg/ml, and another control was set up, that is, mTGF-β1-EXO and 1 μg/ml anti-TGF-β1 mAbs were incubated at 37°C for 1 hour, and FACS was performed three days later. The ratio of CD4 ⁺ Foxp3 ⁺ Tregs was detected.

Results As shown in Figure 10A-B, mTGF-β1-EXO derived from C57BL/6 mice can also significantly inhibit the proliferation of BALB/c mouse T cells and significantly induce the differentiation of Tregs in vitro.

11.2 Second, the safety of reinfusion of allogeneic excretion bodies was evaluated. Each BALB/c mouse was given 10 μg of Control-EXO derived from C57BL/6, injected once every two days through the tail vein, and injected three times in total. Then look around.

In addition, the expression levels of serum IgG and IgE were detected in the first week and the fourth week after Control-EXO injection, and they were all within the normal range. At the same time, the tissue lesions of the main organs of the mice, heart, liver, spleen, lung, and kidney, were also detected in the first week and the fourth week, and there was no difference.

Results As shown in FIG. 10C , there was no significant change in the body weight and behavioral characteristics of the mice. 1 week and 4 weeks after reinfusion of exosomes, the levels of IgE and IgG in the serum of mice did not change significantly. This shows that reinfusion of allogeneic body does not cause significant acute and chronic rejection.

11.3 Treatment of EAE in BALB/c mice with mTGF-β1-EXO derived from C57BL/6 mice

Method: Female C57BL/6 or BALB/C at 6-8 weeks were divided into five groups on average, namely mTGF-β1-EXO, sTGF-β1-EXO, LacZ-EXO, Control-EXO and No treatment group, each group 10 only. 8 days, 5 days and 2 days before modeling, 10 μg/mouse of exosomes derived from C57BL/6 in each group was injected via tail vein, EAE was induced on day 0, scores were recorded, and mTGF-β1-EXO was observed and compared , The preventive effect of sTGF-β1-EXO on EAE. To further test the therapeutic effect of mTGF-β1-EXO and sTGF-β1-EXO on EAE, inject the same dose of exosomes in the tail vein of each group as the prevention group on the 14th, 17th and 21st days of the peak of the onset, and observe the scores.

Results As shown in Figure 10D, mTGF-β1-EXO derived from C57BL/6 mice has a significant therapeutic effect on EAE in BALB/c mice. This indicates that the therapeutic effect of mTGF-β1-EXO on EAE mice is not limited by MHC.

discuss:

An ideal vaccine for widespread use should break through MHC restrictions. Genetically modified DCs can effectively treat autoimmune diseases, but are limited by MHC, which may limit their large-scale application. Experiments have proved that the data effect of the exosomes of the present invention on autoimmune disease models is not limited by MHC,

Compared with imDCs vaccines, exosomes vaccines derived from genetically modified imDCs have the following advantages: (1) Contain proteins related to the special functions of their own cells, and exosomes interact with target cells by carrying immunosuppressive proteins; (2) Strong stability , can be frozen and stored for a long time, eliminating the inconvenience and difficulty of freshly separating and cultivating cells for a long time; (3) non-cellular components, small in size, easy to remove, non-reproductive, weak in antigenicity, small in toxicity and side effects when applied to the human body, and stable efficient.

Therefore, the present invention has broad application prospects in vaccines and treatments of autoimmune diseases.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. a kind of purposes of efflux body, which is characterized in that be used to prepare prevention and/or treat the medicine group of autoimmune disease Object is closed, the autoimmune disease is nervous centralis Demyelination；

Wherein, the efflux body has the feature that

(a) efflux body is the capsule balloon-shaped structure as secreted by immature dendritic cell, the immature dendron shape The modification of film expression cytokines gene of the cell through external source is to express the film expression cytokines；With

(b) efflux body carries the film expression cytokines, wherein the film expression cytokines include TGF-β1。

2. purposes as described in claim 1, which is characterized in that the efflux body diameter is 50-100nm.

3. purposes as described in claim 1, which is characterized in that have and open up on the outer surface of the vesica film of the efflux body Show the film expression cytokines.

4. purposes as described in claim 1, which is characterized in that the film expression cytokines are by film expression type TGF-β 1 Expressed by gene, 1 gene of film expression type TGF-β includes 1 gene of TGF-β being operatively connected with film expression boot sequence, Wherein, the film expression boot sequence includes TM protein sequence and GPI anchorin sequence, also, the TM albumen sequence Column are as shown in SEQ ID NO.:1 or SEQ ID NO.:10；

And the sequence of 1 gene order of TGF-β is as shown in SEQ ID NO.:2.

5. purposes as described in claim 1, which is characterized in that in the immature Dendritic Cells, costimulation point Sub- CD80, CD86 and MHC-II low expression.

6. purposes as claimed in claim 5, which is characterized in that the MHC-II low expression is by the film expression type Caused by cell factor or caused by part.

7. purposes as described in claim 1, which is characterized in that the efflux body also has one or more of feature:

(d) efflux body, which has, inhibits activity that is immune or inhibiting immune system；

(e) the efflux body expression albumen further includes Hsp70, Tsg101 or CD63；

(f) efflux body does not express ER molecular chaperones Grp94.

8. purposes as claimed in claim 1 or 7, which is characterized in that the efflux body has one or more of activity:

(i) cause Treg is grown；

(ii) inhibit the effect of Th1 polarization；

(iii) inhibit Th17 cell differentiation.

9. purposes as described in claim 1, which is characterized in that the nervous centralis Demyelination includes multiple hard Change, experimental autoimmune encephalomyelitis, acute diseminated encephalomyelitis, diffusivity cerebrosclerosis, neuromyelitis optica and Secondary demyelinating disease.

10. purposes as claimed in claim 9, which is characterized in that the secondary demyelinating disease is mainly drawn by systemic disease It rises.