Study Of Mechanism Of Action Memory CD4~+T In Acute Rejection Response After Re-Transplantation

Background:Organ transplantation is now clinically indicated for the treatment of end-stage organ disease,and its use and efficiency are steadily increasing.However,post-transplant rejection of various kinds is still a major cause of loss of function of the transplanted organ and a threat to the life of the transplant recipient.Therefore,some patients may receive a second organ transplant if they are able to do so,but in clinical practice,it is not always possible to perform a second organ transplant in patients who have already undergone a second organ transplant.An organ transplant that,for a variety of reasons,requires a patient to receive another organ transplant because the previous transplanted organ stimulated the recipient to The recipient’s immune system,and has developed corresponding specific antibodies,has an immune memory,and when the organ is transplanted again into the body,the recipient The transplant recipient’s organism will have a more intense acute rejection reaction.And most of the anti-rejection drugs commonly used in clinical practice at this stage are unable to produce good therapeutic effects.And,because of the development of modern medicine,many transplant recipients have often undergone other surgeries and blood transfusions prior to transplantation.as well as special circumstances such as pregnancy,due to exposure to allogeneic tissue,often also leads the recipient of a transplant to certain specific antigens prior to the transplantation procedure The cross-immune effect of developing immune memory also makes rejection more intense even in the first organ transplant.Therefore,how to effectively control acute rejection after retransplantation caused by immune memory is a scientific question that needs to be addressed.Preliminary studies in our laboratory found that memory T cells(T_M)play an important role in the acute rejection response to re-transplantation,that the cytotoxic effects of CD8~+T_M(memory CD8~+T cells),the direct stimulation of the graft by cytokines secreted by CD4~+T_M(memory CD4~+T cells)and the activation of other immune cells resulted in a faster and more intense acute rejection response of the recipient to the transplanted organ and insensitivity to conventional anti-graft rejection drugs.Preliminary studies in our laboratory have screened for key molecules targeting effector memory CD8~+T cells.Therefore,how to suppress the role of memory CD4~+T in the acute rejection response after re-transplantation becomes a challenge that must be overcome.Purpose:We used single-cell sequencing to identify the key gene that plays a role in memory CD4~+T cells in rejection response to re-transplantation,and we used this key gene and the regulation of its codon to effectively inhibit the role of memory CD4~+T cells in acute rejection response after re-transplantation.Methods:1.Construction of a mouse primary skin graft model and a re-skin graft model to stimulate the production of memory CD4~+T cells in mice.2.Splenic lymphocytes were isolated from mouse spleen,and naive CD4~+T cells and memory CD4~+T cells were isolated by magnetic bead sorting.3.Construct a single-cell 3’transcriptome sequencing library using the 10x genomics protocol.Sequence the 10x single-cell library using high-throughput sequencing technology.4.perform bioinformatics analysis of the sequencing results to search for memory CD4~+T cells that may influence the acute rejection after re-transplantation.Key genes of CD4~+T function and possible pathways of action.5.Verify whether regulating the substance encoded by this key gene is effective in suppressing memory CD4~+T in acute rejection after re-transplantation.Cell function and explore possible molecular immunological mechanisms.Results:1.Successful construction of a mouse skin graft model.2.Successful construction of a 10x single-cell sequencing 3’transcriptome library of CD4~+T cells from various groups of mice.3.Bioinformatics analysis revealed that the key gene for the role of memory CD4~+T cells in the acute rejection response after re-transplantation is ctsb,which encodes histone B.4.It was found that the key gene ctsb and another memory CD4+T cell highly expressed gene cd74enable CD4~+T cells to function as secretory cytokines that activate other immune cells through the Antigen processing and presentation pathway.Conclusions:In this experiment,we isolated mouse naive CD4~+T cells and memory CD4~+T cells by successfully establishing a mouse reskin graft model,and using 10x genomics single-cell sequencing technology,a 3’end transcriptome library was constructed and sequenced.after bioinformatics analysis,we revealed that memory CD4~+T cells are involved in rejection function after re-transplantation.The key gene is ctsb,encodes cathepsin B.And together with cd74 and other genes through Antigen processing and presentation pathway,CD4~+T cells activated by MHC class II molecules,promoting their secretion of cytokines,as well as having an activating effect on other immune cells.Antagonism of ctsb-encoded cathepsin B is expected to reduce the acute rejection of re-transplantation involving memory CD4~+T cells.,and also for organ transplantation in patients with malignant tumors,antagonizing cathepsin B can reduce malignant tumor recurrence and translocation,and because memory CD4+T cells also overexpress complement-related genes,there is a corresponding reduction in complement harm from antibody-related rejection brought about by activation of the pathway,as well as mitigating the extent of subsequent chronic rejection that may occur.