Method for reducing or eliminating allograft rejection by using thymus vaccine

Abstract

Provided is a method for reducing or eliminating allograft rejection by using a thymus vaccine. The method comprises the following steps: expressing major histocompatibility complex (MHC) from a donor and / or grafting a thymus epithelial cell from a donor in the thymus tissue of a recipient by means of the thymus vaccine. The thymus vaccine includes a thymic gene vaccine and / or a thymocyte vaccine. The thymic gene vaccine is used for expressing an MHC antigen from the donor in the thymus of the recipient, and comprises: (1) a gene expression vector; and (2) polynucleotides separately encoding MHC class I molecules and MHC class II molecules of the donor. The thymocyte vaccine is prepared by the following steps: sorting by using an anti-Epcam antibody to give thymus epithelial cells. By establishing a convenient donor source, the limitations of allograft caused by MHC mismatch are overcome, thus helping solve the clinical problem of limited donor sources.

Description

A method for reducing or eliminating allogeneic transplant rejection using thymus vaccine This application claims priority to Chinese patent application No. 2024101713279, filed on February 6, 2024. This application incorporates the entire text of the aforementioned Chinese patent application. Technical Field The present invention belongs to the field of biotechnology, and specifically relates to a method for reducing or eliminating allogeneic transplant rejection by utilizing a thymus vaccine, the thymus vaccine (including a thymus gene vaccine or a thymus cell vaccine) used in the method, and applications thereof. Background Art Cell organ transplantation is considered the ultimate treatment for many organic diseases. For example, hematopoietic stem cell transplantation is used to treat blood diseases, and allogeneic organ transplantation or xenogeneic organ transplantation is used to treat organ failure, necrosis and other diseases. Every year, more than 140,000 patients receive organ transplants worldwide, mainly including kidneys, livers, hearts, lungs, pancreas and small intestines. China's annual demand for transplanted organs exceeds 300,000. However, the proportion of people who meet the conditions and receive organ transplants does not exceed 10%. This situation is faced worldwide. The main reasons for this situation are organ shortages and immune rejection reactions. Solving the current organ shortage situation can be improved through more donations, but immune rejection reactions have not yet been completely resolved. [1-2] . When an allogeneic transplant is performed, due to the high polymorphism of MHC, if the MHC of the donor and recipient do not match, the recipient's immune system will recognize the transplant as "non-self" and launch an attack, leading to rejection. [3-4] The main function of MHC is to present antigens to T cells so that they can recognize and attack "non-self" cells. The T cell's recognition mechanism for "self" and "non-self" is formed in the thymus through positive and negative selection. [5-6] MHC plays a key role in this selection process. In the thymus, the interaction between MHC and T cells determines the fate of T cells. [7] In positive selection, if the affinity between the T cell receptor (TCR) and the MHC molecule is too low, the T cell will die due to neglect; in negative selection, if the affinity is too high, the T cell will also die, avoiding autoimmunity. Mature T cells that have undergone positive and negative selection will form tolerance to "self". [8] . Alternative therapies of pluripotent stem cells and organ transplantation are considered the ultimate treatment for many organic diseases. However, due to the high polymorphism of MHC, when an allogeneic transplant is performed, if the MHC of the donor and recipient do not match, the recipient's immune system will recognize the transplant as "non-self" and launch an attack, leading to rejection. The problem of transplant rejection caused by MHC mismatch is still a major challenge. Although researchers have proposed tolerance strategies such as mixed hematopoietic chimeras, there is still a risk of tolerance instability and severe graft-versus-host disease (GvHD). [9-12] Therefore, it is urgent to develop a strategy that can transcend the MHC matching restriction and achieve a broad range of immune tolerance. Although the main factor causing allogeneic transplant rejection is MHC mismatch, the presence of minor histocompatibility antigens may also lead to Therefore, it is of great significance to develop a strategy that perfectly solves the MHC matching restriction and adoptive transfer of minor histocompatibility antigens. Gene therapy (GT) can precisely target specific genes for repair or regulation through the use of specific vectors, thereby changing the expression pattern of cells to achieve therapeutic purposes. Adeno-associated virus (AAV) vectors have been successfully used in gene therapy applications. In particular, gene therapy in the thymus provides an opportunity to directly intervene in the development of T cells. For example, after Pouzolles et al. injected AAV-ZAP-70 intrathymically (IT) into the thymus of ZAP-70-deficient mice, they found rapid development of functional T lymphocytes and rapid reconstruction of the thymic medulla. This fully demonstrates the great potential and advantages of gene therapy in the treatment of refractory diseases. Technological advances in gene therapy provide new ideas for solving the problem of transplant rejection. Using gene expression technology, the polymorphism of donor-specific antigens makes it difficult to express all donor antigens in the recipient thymus. Therefore, developing technologies that can express all donor-specific antigens in recipient thymic cells and achieve perfect adoptive transfer of donor antigens into the recipient thymus is key to resolving immune rejection during organ transplantation and cell therapy. In thymic tissue, the main cells involved in T cell development include thymic epithelial cells and dendritic cells. The expression of dendritic cell thymic tissue antigens in the recipient thymus is a key solution to the problem of T cell education in the thymus, and cell transplantation is an optimized solution. The current effective strategy to solve MHC matching is to use immunosuppressants in combination with organ transplantation on the basis of meeting the matching requirements as much as possible. At the same time, scientists have reduced the donor's immune rejection of recipient cells by establishing low-immunogenicity donor cells (Publication No.: TW202321435A, Publication No.: CN115335087A). Based on the transplant rejection problem caused by MHC mismatch, the development of an immune tolerance strategy that can transcend MHC matching restrictions and achieve broad applicability is the key to solving transplant rejection. In the patent with Publication No.: CN112074277A, the technology is based on the culture and transplantation of allogeneic thymic tissue to achieve donor-specific tolerance of allogeneic organ transplants. Summary of the Invention To solve the problem in the prior art that allogeneic transplantation has great limitations on donors, the present invention provides a method for reducing or eliminating allogeneic transplant rejection using a thymus vaccine, a thymus vaccine (including a thymus gene vaccine and a thymus cell vaccine) used in the method, and its application. Existing technology cultivates donor-derived thymic tissue and transplants it into the muscle to form a new simulated thymus in order to achieve specific tolerance to the donor-derived transplant. This technology has great limitations on the donor. T cells undergo a complex process of maturation in the thymus, mainly including negative selection and positive selection, to delete self-reactive T cells. Mature T cells that have undergone the selection process achieve immune tolerance to themselves. This selection process is mainly determined by self-antigens. So when T cells are stimulated by foreign antigens during the selection process, will the T cells that are reactive to foreign antigens be Deletion after positive-negative selection, that is, whether T cells can be generated that do not attack cells derived from the corresponding antigen, is a key issue facing allogeneic transplantation. The present invention mimics the process by which natural T cells develop self-tolerance, using a low-immunogenic AAV vector to express the donor's allogeneic MHC (allo-MHC) in the recipient's thymus. After positive and negative selection, donor-reactive T cells undergo apoptosis, and mature T cells recognize cells expressing both autologous and allogeneic MHC as "self," establishing long-term, stable donor-specific tolerance. Furthermore, the key to the success of allogeneic cell or organ transplantation is tissue compatibility, and the factor that determines tissue compatibility is histocompatibility antigens. Histocompatibility antigens are divided into major histocompatibility antigens and minor histocompatibility antigens. Antigens that cause acute rejection reactions are called major histocompatibility antigen mismatches; however, immune rejection still occurs when some identical twins with the same major histocompatibility antigens undergo organ transplantation, and the main reason is minor histocompatibility antigen mismatches. The thymic gene vaccine technology solution is to express the donor's major histocompatibility antigens in thymic epithelial cells, which can solve the immune rejection problem during cell or organ transplantation. However, this solution is still not perfect when encountering immune rejection reactions caused by minor histocompatibility antigens. Therefore, the present invention proposes for the first time a method for co-expressing a donor's major and minor histocompatibility antigens in the recipient's thymus. While the polymorphism of minor histocompatibility antigens currently prevents their expression via transgenic technology, transplanting donor-derived thymic epithelial cells into the recipient's thymus allows for the complete expression of donor histocompatibility antigens within the recipient's thymus. To address the limitations of MHC matching and the polymorphism of adoptively transferred minor histocompatibility antigens, the present invention has developed a thymic vaccine technology solution for adoptively transferring donor antigens into the recipient thymus, which includes: 1) expressing the donor MHC in the recipient thymus through adenovirus infection; 2) transplanting donor thymic epithelial cells into the recipient thymus to achieve expression of the donor MHC and minor histocompatibility antigens in the recipient thymus. Therefore, one of the technical solutions of the present invention provides a method for reducing or eliminating allogeneic transplant rejection, wherein the method comprises the following steps: The donor's MHC is expressed in the recipient's thymus tissue and / or the donor's thymic epithelial cells are transplanted through thymus vaccines; the thymus vaccines include thymus gene vaccines and / or thymocyte vaccines. Preferably, the method is for non-therapeutic purposes; and / or, the method is performed in vitro or ex vivo. In some preferred embodiments, the expression of the donor MHC is achieved by injecting a thymic gene vaccine into the recipient's thymus tissue. Preferably, the thymus gene vaccine comprises: (1) Gene expression vector; (2) polynucleotides encoding the donor's MHC class I and MHC class II molecules, respectively; The polynucleotide is operably linked to the gene expression vector, and the MHC background of the donor is different from the MHC background of the recipient. More preferably, the gene expression vector includes a plasmid vector and a viral vector; and / or, the polynucleotides encoding the donor's MHC class I molecule and MHC class II molecule are located in the same gene expression vector or in two separate gene expression vectors. In some preferred embodiments, the viral vector is a lentiviral vector or an adeno-associated viral vector (AAV), such as AAV2 / 8. Preferably, the thymus gene vaccine comprises: AAV-CMV-H2-Kbαchain-IRES-H2-Dbαchain and AAV-CMV-I-Abαchain-IRES-βchain. More preferably, the thymus gene vaccine comprises: isolated nucleic acids having nucleotide sequences as shown in SEQ ID NO: 1 and 2, respectively; and / or, the thymus gene vaccine is injected into 1-5 sites, for example, 3 sites, of the left and right lobes of the recipient's thymus. In some more preferred embodiments, after the injection of the thymic gene vaccine, the recipient is acted upon by the thymic vaccine, and newly mature T cells in the thymus are educated by both the recipient's own MHC and the MHC expressed by the thymic vaccine, thereby achieving deletion of donor-reactive T cells. Preferably, before injecting the thymus vaccine, the following steps are also included: (i) injection or no injection of IFN-γ; (ii) injection of T cell depleting agents or immunosuppressants; the T cell depleting agents are, for example, antibodies 2.43 and GK1.5; (iii) Irradiation; for example, 1.5-3 Gy irradiation for 1-2 minutes on the second day. In this study, IFN-γ was shown to stimulate rapid and high expression of MHC in mice. However, in human recipients, IFN-γ injection is unnecessary because the body can produce IFN-γ on its own. This article demonstrates T cell depletion in mice. Immunosuppressants are used as a basic treatment in human organ transplantation and can effectively prevent rejection. Ideally, immunosuppressant therapy would prevent graft rejection while minimizing toxic side effects. Commonly used immunosuppressants include azathioprine, mycophenolate mofetil, corticosteroids, cyclosporine, tacrolimus, antilymphocyte globulin, and moneromonas-CD3. Therefore, the second technical solution of the present invention provides a method for tissue or organ transplantation, which comprises the following steps: A. using the method described in one of the technical solutions to reduce or eliminate allogeneic transplant rejection; B. Cell, tissue, or organ transplantation before or after the recipient's peripheral blood T cells return to normal levels; Preferably, the tissue or organ is selected from one or more of skin, pancreatic islets, nerves, kidneys, liver, heart, small intestine, fat and bones. The thymus vaccine technology of the present invention includes two main aspects: one is to express the donor MHC in the recipient's thymus based on gene editing technology to solve the problem of MHC matching restriction. This strategy based on gene therapy has opened up a new path for the application of gene therapy in the field of transplant immunity. Current gene therapy has shown great potential in the treatment of many diseases. Gene therapy is to introduce specific genes into cells by using specific vectors (such as adeno-associated virus, AAV), thereby changing their expression pattern and achieving the purpose of treatment. The second is to transplant allogeneic thymic epithelial cells in the recipient's thymus. The thymic epithelial cells of this donor origin carry all the antigen information of the donor, which not only solves the MHC mismatch but also avoids the immune rejection that may be caused by minor tissue compatibility antigens. Therefore, the third technical solution of the present invention provides a thymic gene vaccine, which is used to express donor histocompatibility antigens (MHC) in the recipient thymus and comprises: (1) Gene expression vector; (2) polynucleotides encoding the donor's MHC class I and MHC class II molecules, respectively; The polynucleotide is operably linked to the gene expression vector, and the MHC background of the donor is different from the MHC background of the recipient. Preferably, the gene expression vector includes a plasmid vector and a viral vector; and / or, the polynucleotides encoding the donor's MHC class I molecule and MHC class II molecule are located in the same gene expression vector or in separate gene expression vectors. In some preferred embodiments, the viral vector is a lentiviral vector or an adeno-associated viral vector (AAV), such as AAV2 / 8. Preferably, the thymus gene vaccine comprises: AAV-CMV-H2-Kbαchain-IRES-H2-Dbαchain and AAV-CMV-I-Abαchain-IRES-βchain. More preferably, the thymus gene vaccine comprises: isolated nucleic acids whose nucleotide sequences are shown as SEQ ID NOs: 1 and 2, respectively. A fourth technical solution of the present invention provides a thymocyte vaccine, which is prepared by the following steps: thymic epithelial cells are obtained by sorting using anti-Epcam antibodies. Preferably, the dose of the thymic epithelial cells is 2 million per recipient; and / or, the thymic epithelial cells are suspended in phosphate buffered saline (PBS) to prepare the thymic cell vaccine. When the recipient is human, the amount of thymic epithelial cells transplanted is approximately 50% of the total amount of human thymocytes based on the mouse ratio. The present invention sorts the donor thymic epithelial cells and transplants them into the recipient's thymus. According to the present invention, those skilled in the art can transplant the donor thymic epithelial cells obtained in any form (such as primary cells, pluripotent stem cell differentiation) into the recipient. Thymus, can achieve the effect of resolving immune rejection. A fifth technical solution of the present invention provides a thymocyte vaccine kit, which includes the thymus gene vaccine as described in the third technical solution or the thymocyte vaccine as described in the fourth technical solution; Optionally, further comprising (i) IFN-γ; and / or (ii) T cell depleting agents or immunosuppressants. Technical solution six of the present invention provides a thymus gene vaccine as described in technical solution three, a thymus cell vaccine as described in technical solution four, or a thymus cell vaccine kit as described in technical solution five for use in preparing an immune preparation for reducing or eliminating allogeneic transplant rejection or in reducing or eliminating allogeneic transplant rejection. On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention. The reagents and raw materials used in the present invention are commercially available. The positive progress effect of the present invention is: This invention develops a transplant tolerance strategy based on a thymus vaccine, establishing a convenient and universal donor source. This overcomes the limitations of allogeneic transplantation due to MHC mismatches and helps resolve the donor source issue in clinical practice. This gene therapy-based strategy opens new avenues for the application of gene therapy in transplant immunity. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram of the plasmid; A is the map of the construction of the MHCβ chain of the thymus gene vaccine donor, which is connected to the H2 on the donor MHC chain β on the PGMAAV-4878 expression vector. Kb and H2 Db Gene segment; B is the map of the construction of the donor MHCα chain thymic gene vaccine, which is connected to the donor MHCα chain Iα (IA) gene segment on the PGMAAV-4878 expression vector. FIG2 shows the expression of thymic gene vaccine in thymic epithelial cells of recipients. FIG3 shows the expression of thymic gene vaccine in dendritic cells in the thymus of recipients. FIG4 shows the deletion of donor reactive T cells under the action of thymic gene vaccine. Figure 5 shows the results of donor skin transplantation under the action of thymus gene vaccine. Figure 6 shows that donor islet transplantation under the action of thymus gene vaccine can effectively treat type 1 diabetes. FIG7 shows teratoma formation in allogeneic mice under the action of thymic gene vaccine. Figure 8 shows the growth of multiple organ tissues under the action of thymus gene vaccine. Figure 9 shows the thymocyte vaccine sorting results. FIG10 shows the results of long-term survival of thymocyte vaccine in the thymus of recipients. Figure 11 shows the results of donor skin transplantation under the action of thymocyte vaccine. Figure 12 shows that donor islet transplantation under the action of thymocyte vaccine can effectively treat type 1 diabetes. DETAILED DESCRIPTION The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications. Example 1 Thymus gene vaccine To achieve allo-MHC expression in the recipient thymus, a recombinant AAV virus was constructed based on the gene sequence expressing the donor MHC. First, a pair of mice with completely mismatched MHC were selected, with the recipient Balb / c mouse having an MHC background of H2d and the donor C57 mouse having an MHC background of H2b. Next, based on the MHC gene sequence of the donor mouse, the present invention constructed recombinant AAV viruses expressing H2b class I and class II molecules, namely: AAV2 / 8-CMV-H2-Kbαchain-IRES-H2-Dbαchain and AAV2 / 8-CMV-I-Abαchain-IRES-βchain (collectively referred to as thymic gene vaccines). The plasmid schematics are shown in Figure 1 A and B, respectively. The corresponding nucleotide sequences are named GPAAV-CMV-H2-Kb-IRES-H2-Db-WPRE and GPAAV-CMV-I-Ab-alpha-IRES-beta-WPRE, respectively. Nucleotide sequence of GPAAV-CMV-H2-Kb-IRES-H2-Db-WPRE Nucleotide sequence of GPAAV-CMV-I-Abalpha-IRES-beta-WPRE First, 8-week-old Balb / C mice were intraperitoneally injected with 400 μl of a T cell-depleting antibody cocktail consisting of antibodies 2.43 (bioxcell, CAT: BE0061) and GK1.5 (bioxcell, CAT: BP0003-1). GK1.5 was administered at a concentration of 9.79 mg / ml in 40.8 μl / vial, while 2.43 was administered at a concentration of 9.9 mg / ml in 40.4 μl / vial. On the second day, T cell depletion was performed by irradiation at a strength of 1.5 Gry for 1 minute. One week later, the thymic gene vaccine was injected into the thymus of the T cell-depleted Balb / C mice at three sites each in the upper, middle, and lower lobes. Following successful vaccination, newly mature T cells in the recipient mice's thymus were educated through both their own MHC and the MHC expressed by the thymic gene vaccine, resulting in the elimination of donor-reactive T cells. After 42 days, when the peripheral blood T cells of the recipient mice returned to normal levels, experiments such as skin, pancreatic islet, kidney, liver and heart transplantation from C57 mice were performed. Four weeks after the skin transplant, peripheral blood was collected from the recipient mice, and CD3-positive T cells were sorted by flow cytometry for scRNAseq and TCRseq. Single-cell sequencing experiments demonstrated that the technology of this invention achieved clonal elimination of donor-reactive T cells, educated these T cells to no longer attack the recipient. After the donor thymic gene vaccine was injected into the recipient's thymus, flow cytometry was used to measure the efficiency of the thymic gene vaccine infection in the recipient's thymic epithelial cells. Thymic epithelial cells infected with the thymic gene vaccine expressed the donor's MHC for a long time, maintaining high levels of expression on day 90 (see Figure 2, A and B). After the donor thymus gene vaccine is injected into the recipient's thymus, the efficiency of thymus gene vaccine infection of the recipient's thymus dendritic cells is detected by flow cytometry. After the thymus dendritic cells are infected with the thymus gene vaccine, they express the donor's MHC for a long time. Although the expression level decreased slightly, it still maintained a high level of expression on day 90. The decrease in expression level may be due to the mobility of dendritic cells (see Figure 3A and B). After thymic gene vaccine injection, newly matured T cells are tolerant to both the recipient and the donor. Single-cell TCR sequencing was used to analyze TCR expression in newly mature T cells. Using TRBV5, TRBV19, TRBV13-2, TRBV31, TRBV20, and TRBV1, which are highly expressed, as examples, the results showed that after thymic gene vaccine administration, the expression levels of the donor-reactive TCRs TRBV31, TRBV20, and TRBV1 decreased, and the donor-reactive T cells were deleted (see Figure 4, A and B). After the thymus gene vaccine takes effect, the donor's reactive T cells are deleted, and the recipient's newly mature T cells do not attack the cells, tissues and organs of the donor. The skin, which is the most difficult to transplant, was selected to verify the effect of the vaccine. The results showed that the donor skin survived well after being transplanted to the recipient (see Figure 5); among them, "PTD0" and "PTD0" in the figure represent the 0th day and the 21st day after transplantation, respectively. Type 1 diabetes was established in wild-type C57 mice, wild-type Balb / c mice, and Balb / c mice treated with a thymic gene vaccine. After successful diabetic mouse models, pancreatic islets derived from C57 mice were transplanted under the renal capsule of these three diabetic mouse models, and blood glucose levels were measured at various time points. After autologous C57 islet transplantation (Auto), blood glucose levels in these models decreased significantly and returned to normal within three weeks. After the fourth week, blood glucose levels remained elevated, indicating functional depletion of the transplanted islets due to excessive insulin production. In mice receiving the empty AAV virus vaccine (AAV Con) injected into the thymus, blood glucose levels decreased slightly within two weeks after islet transplantation, reflecting the limited effect of the transplanted islets before they are completely rejected. In mice receiving the thymic gene vaccine (TV), blood glucose levels after islet transplantation were similar to those in mice receiving autologous islet transplantation. These results demonstrate that allogeneic islet transplantation, under the influence of a thymic gene vaccine, is an effective treatment for type 1 diabetes, as shown in Figure 6. To further validate the allogeneic effect, teratoma formation experiments were conducted using subcutaneous injections of C57 mouse embryonic stem cells in C57 mice, Balb / c mice, C3H mice, and Balb / c mice injected with a thymic gene vaccine. Teratomas developed successfully in the C57 mouse group (Auto con) and the Balb / c mouse group (TV) injected with a thymic gene vaccine, with no significant difference in teratoma size. Teratomas did not develop in the Balb / c mouse group (Allo con) or the C3H mouse group (3rd con), as shown in Figure 7A and B. Subcutaneous transplantation of pluripotent stem cells into the C57 mouse group (Auto con) and the Balb / c mouse group (TV) injected with the thymus gene vaccine successfully grew a variety of organ tissues, indicating that the thymus gene vaccine has a good effect on the transplantation and growth of allogeneic multiple organs, proving that the thymus gene vaccine can effectively solve the allogeneic transplantation of multiple organs, as shown in Figure 8. Example 2 Thymocyte vaccine C57BL / 6JSmoc-Gt(ROSA)26S or em1(CAG-Luc-EGFP)Smoc male mice were mated with female mice. Newborn mice P1-P7 were obtained and subjected to magnetic bead sorting. Thymic epithelial cells were then sorted using anti-Epcam mAb to obtain a thymocyte vaccine, as shown in Figure 9. Thymic epithelial cells were sorted from the thymus of newborn C57 mice P1-7 using the CD326 surface marker. Thymic epithelial cells were suspended in PBS containing matrigel + 10% FBS and transplanted into the left and right lobes of the thymus of recipient mice that had undergone T cell depletion (same as in Example 1) at a rate of 2 million thymic epithelial cells per mouse. One million cells were suspended in 5 μl of PBS buffer at each site for injection. Luciferase fluorescence detection of the donor thymic epithelial cells transplanted into the recipient thymus revealed that they were still present 90 days after transplantation. After the thymocyte vaccine is successfully obtained, it is transplanted into the recipient's thymus. An isotype control and a control without prior T cell depletion are set up. The results show that the thymocyte vaccine survives in the recipient's thymus for a long time after transplantation (see Figure 10). After the thymocyte vaccine was administered, the effect of the thymocyte vaccine was verified using skin that was the most difficult to transplant. The results showed that after the thymocyte vaccine was administered, the donor skin survived well after being transplanted to the recipient (see Figure 11); in the figure, "PTD0" and "PTD0" represent the 0th day and the 21st day after transplantation, respectively. Type 1 diabetes was established in wild-type C57 mice, wild-type Balb / c mice undergoing sham surgery, and Balb / c mice treated with a thymocyte vaccine. After successful diabetic mouse models, pancreatic islets derived from C57 mice were transplanted under the renal capsule of these three diabetic mouse models, and blood glucose levels were measured at various time points. After autologous C57 islet transplantation (Auto), blood glucose levels in these mice decreased significantly and returned to normal within three weeks. After the fourth week, blood glucose levels continued to rise, driven by excessive insulin production by the transplanted islets, leading to functional exhaustion. In the sham thymocyte surgery group (Blank), blood glucose levels decreased slightly within two weeks after islet transplantation, driven by the minimal effect of the transplanted islets before they were completely rejected. Blood glucose levels in mice receiving the thymocyte vaccine (AlloTEC) were similar to those in autologous islet transplantation. These results demonstrate that allogeneic islet transplantation, under the influence of a thymocyte vaccine, is an effective treatment for type 1 diabetes, as shown in Figure 12. References [1]Global activity in organ transplantation estimates. [2] China Organ Transplant Development Report (book) [3]Montgomery,R.A.,Tatapudi,V.S.,Leffell,M.S.&Zachary,A.A.HLA in transplantation.Nat Rev Nephrol 14,558-570,doi:10.1038 / s41581-018-0039-x(2018). [4]Bodmer,J.G.et al.Nomenclature for factors of the HLA system,1998.Tissue Antigens 53,407-446,doi:10.1034 / j.1399-0039.1999.530421.x(1999). [5]Zachary,A.A.&Leffell,M.S.HLA Mismatching Strategies for Solid Organ Transplantation-A Balancing Act.Front Immunol 7,575,doi:10.3389 / fimmu.2016.00575(2016). [6]Williams,R.C.,Opelz,G.,McGarvey,C.J.,Weil,E.J.&Chakkera,H.A.The Risk of Transplant Failure With HLA Mismatch in First Adult Kidney Allografts From Deceased Donors.Transplantation 100,1094-1102,doi:10.1097 / TP.0000000000001115(2016). [7]Jameson,S.C.,Hogquist,K.A.&Bevan,M.J.Positive selection of thymocytes.Annu Rev Immunol 13,93-126,doi:10.1146 / annurev.iy.13.040195.000521(1995). [8] Goldrath, AW & Bevan, MJ Selecting and maintaining a diverse T-cell repertoire. Nature402, 255-262, doi:10.1038 / 46218(1999). [9] Cobbold, SP, Martin, G., Qin, S. & Waldmann, H. Monoclonal antibodies to promote marrow engraftment and tissue graft tolerance. Nature 323, 164-166, doi: 10.1038 / 323164a0 (1986).

[0010] Pilat, N. & Wekerle, T. Transplantation tolerance through mixed chimerism. Nat Rev Nephrol 6, 594-605, doi:10.1038 / nrneph.2010.110(2010).

[0011] Kawai,T.et al.HLA-mismatched renal transplantation without maintenance immunosuppression.N Engl J Med 358,353-361,doi:10.1056 / NEJMoa071074(2008).

[0012] Blazar, BR, Murphy, WJ & Abedi, M. Advances in graft-versus-host disease biology and therapy. Nat Rev Immunol 12, 443-458, doi:10.1038 / nri3212 (2012). Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for reducing or eliminating allogeneic transplant rejection, characterized in that: The method comprises the following steps: Expressing the donor's MHC in the recipient's thymic tissue and / or transplanting the donor's thymic epithelial cells through a thymic vaccine; the thymic vaccine includes a thymic gene vaccine and / or a thymic cell vaccine; Preferably, the method is for non-therapeutic purposes; and / or, the method is performed in vitro or ex vivo.

2. The method according to claim 1, wherein The expression of the donor MHC is achieved by injecting a thymic gene vaccine into the recipient's thymus tissue; Preferably, the thymus gene vaccine comprises: (1) Gene expression vector; (2) polynucleotides encoding the donor's MHC class I and MHC class II molecules, respectively; wherein the polynucleotide is operably linked to the gene expression vector, and the MHC background of the donor is different from the MHC background of the recipient; More preferably, the gene expression vector includes a plasmid vector, a phage vector, a non-viral vector and a viral vector; and / or, the polynucleotides encoding the donor's MHC class I molecule and MHC class II molecule are located in the same gene expression vector or in separate gene expression vectors.

3. The method according to claim 2, wherein The viral vector is a lentiviral vector or an adeno-associated viral vector (AAV), such as AAV2 / 8; Preferably, the thymic gene vaccine comprises: AAV-CMV-H2-Kbαchain-IRES-H2-Dbαchain and AAV-CMV-I-Abαchain-IRES-βchain; More preferably, the thymus gene vaccine comprises: isolated nucleic acids having nucleotide sequences as shown in SEQ ID NO: 1 and 2, respectively; and / or, the thymus gene vaccine is injected into 1-5 sites, for example, 3 sites, of the left and right lobes of the recipient's thymus.

4. The method according to claim 1, wherein The preparation of the thymocyte vaccine comprises the following steps: using anti-Epcam antibodies to sort and obtain thymic epithelial cells; Preferably, the thymocyte vaccine is transplanted into 1-3 sites in each of the left and right lobes of the recipient's thymus; and / or the dose of the thymocyte vaccine is 2 million thymic epithelial cells per recipient; More preferably, the thymic epithelial cells are suspended in phosphate buffered saline (PBS) to prepare the thymocyte vaccine.

5. The method according to claim 2 and / or claim 4, wherein: After thymic vaccine administration, deletion of donor-reactive T cells is achieved in newly mature T cells; Preferably, before injecting the thymus vaccine, the following steps are also included: (i) injection or no injection of IFN-γ; (ii) injection of T cell depleting agents or immunosuppressants; the T cell depleting agents are, for example, antibodies 2.43 and GK1.5; (iii) Irradiation; for example, 1.5-3 Gy irradiation for 1-2 minutes on the second day.

6. A method for tissue or organ transplantation, characterized in that: It includes the following steps: A. reducing or eliminating allogeneic transplant rejection using the method according to any one of claims 1 to 5; B. Cell, tissue, or organ transplantation before or after the recipient's peripheral blood T cells return to normal levels; Preferably, the tissue or organ is selected from one or more of skin, pancreatic islets, nerves, kidneys, liver, heart, small intestine, fat and bones.

7. A thymus gene vaccine, characterized in that: The thymic gene vaccine is used to express donor histocompatibility antigens (MHC) in the recipient's thymus and includes: (1) Gene expression vector; (2) polynucleotides encoding the donor's MHC class I and MHC class II molecules, respectively; wherein the polynucleotide is operably linked to the gene expression vector, and the MHC background of the donor is different from the MHC background of the recipient; Preferably, the gene expression vector includes a plasmid vector and a viral vector; and / or, the polynucleotides encoding the donor's MHC class I molecule and MHC class II molecule are located in the same gene expression vector or in two separate gene expression vectors.

8. The thymus gene vaccine according to claim 7, characterized in that The viral vector is a lentiviral vector or an adeno-associated viral vector (AAV), such as AAV2 / 8; Preferably, the thymic gene vaccine comprises: AAV-CMV-H2-Kbαchain-IRES-H2-Dbαchain and AAV-CMV-I-Abαchain-IRES-βchain; More preferably, the thymus gene vaccine comprises: isolated nucleic acids whose nucleotide sequences are shown as SEQ ID NOs: 1 and 2, respectively.

9. A thymocyte vaccine, characterized in that The method is prepared by the following steps: thymic epithelial cells are obtained by sorting using anti-Epcam antibodies; Preferably, the dose of the thymic epithelial cells is 2 million per recipient; and / or, the thymic epithelial cells are suspended in phosphate buffered saline (PBS) to prepare the thymic cell vaccine.

10. A thymocyte vaccine kit, characterized in that: It includes the thymic gene vaccine according to claim 7 or 8, or the thymocyte vaccine according to claim 9; Optionally, further comprising (i) IFN-γ; and / or (ii) T cell depleting agents or immunosuppressants.

11. Use of the thymus gene vaccine according to claim 7 or 8, the thymocyte vaccine according to claim 9, or the thymocyte vaccine kit according to claim 10 in preparing an immune preparation for reducing or eliminating allograft rejection or in reducing or eliminating allograft rejection.

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