Study On Key Molecules And Antagonists Of Acute Rejection Of Retransplantation Induced By Memory CD8~+ T Cells

Organ transplantation is called"the trick of modern medicine"and is an effective means for clinical treatment of end-stage organ diseases.In2018,China carried out 20,200 organ transplant operations,an increase of21%over 2017,ranking sond in the world.However,transplant rejection is still an important cause of loss of transplant organ function,so a considerable number of patients have to undergo organ transplantation again,and acute rejection after retransplantation will occur earlier and more intense,and no anti-reorganism has been found yet.An effective drug for retransplant rejection is urgently needed.In clinical practice,it is found that in patients who need to be transplanted again after the first transplant failure,the immune system has immune memory in advance for certain specific antigens produced after the first transplant of the organ,and after the transplanted organ enters the body again,the body is in a short time.Acute rejection of the transplanted organ can occur and is more intense than the first rejection and is not sensitive to the anti-rejection drugs currently used clinically.Theref ore,how to effectively control the re-transplantation acute rejection caused by immune memory is a scientific problem to be solved urgently.Previous studies in our laboratory found that effector memory CD8+T cells(CD8⁺T_EM)resulted in a faster,more intense receptor rejection of the donor（transplanted organ）and was not sensitive to the transplant rejection drug cyclosporine A.Therefore,how to inhibit CD8⁺T_EM-induced re-transplantation of acute rejection is a difficult problem to overcome.Based on this,we first used single-cell sequencing technology to find key molecules that can effectively inhibit CD8⁺T_EM-induced acute rejection of re-transplantation.Then,it is verified whether the key molecule can exert an ideal inhibitory effect on the prol iferation and activity of CD8⁺T_EM and explore its mechanism of action,and complete related experiments and studies in vitro.Finally,by constructing an allogeneic mouse organ（heart）retransplantation model,it was demonstrated in vivo whether this key molecule can be a key target for effective inhibition of CD8⁺T_EM-mediated acute rejection of organ transplantation.The main research contents and results of this topic are as follows:Objective:By exploring the key molecules of effector memory CD8⁺T cells,effective inhibition of acute rejection of re-organ transplantation caused by effector memory CD8⁺T cells was achieved.Methods:1.The single-cell transcriptome sequencing library was constructed using the Smart-seq2 protocol,and the c DNA library was sequenced by high-throughput sequencing technology.The bioinformatics analysis of the sequencing results was carried out to find the key molecule to effectively inhibit the acute rejection of re-transplantation caused by CD8⁺T_EM.2.Single cell sequencing results were verified by q PCR,WB,ELISA,etc.The inhibitory effect of this key molecule on the proliferation and activity of CD8⁺T_EM was verified by CFSE staining and ELISA.The PI3K-Akt signaling pathway was detected by WB at 0 min,2 min,5 min,10 min,15 min,and 30 min,respectively,by blocking the Integrinα1（itga1）and PI3K sites of CD8⁺T_EM by using itga1 monoclonal antibody and PI3K small molecule blocker.The expression of downstream proteins p-PI3K/PI3K,p-Akt/Akt,p-p21/p21,p-p27/p27,and CDK6 was studied,and the mechanism of action of itga1 was studied to complete related experiments and studies in vitro.3.Construction of an allogeneic mouse re-organ（heart）transplantation model by observing the survival time of the transplanted heart,the histopathological grade of acute rejection of the transplanted heart,and the Chemokine C-C motif ligand 3（CCL3）in the recipient mouse,the Chemokine C-C motif ligand 3（CCL4）,Interferon-γ（IFN-γ）concentration,the distribution CD8⁺T_EM transplantation into the heart and spleen of recipient mice,and other indicators in vivo to verify whether the key molecule can effectively inhibit CD8⁺T_EM-mediated re-transplantation of acute rejection and explore its possible molecular immunological mechanisms.Result:1.Single cell sequencing results showed that itga1,CCL3,CCL4 and IFN-γwere highly expressed on CD8⁺T_EM,and itga1 exerted its regulation on CD8⁺T_EM proliferation activity through PI3K-Akt signaling pathway.2.q PCR results showed that the expression level of itga1 in CD8⁺T_EMwas significantly higher than that of na?ve CD8⁺T cells(CD8⁺T_NA)（P<0.0001）and central memory CD8⁺T cells(CD8⁺T_CM)（P<0.0001）.The expression of CCL3 in CD8⁺T_EM was significantly higher than that of CD8⁺T_NA（P<0.0001）and CD8⁺T_CM（P<0.01）.The expression of CCL4 in CD8⁺T_EM was significantly higher than that of CD8⁺T_NA（P<0.001）and CD8⁺T_CM（P<0.05）.The expression of IFN-γin CD8⁺T_EM was significantly higher than that of CD8⁺T_NA（P<0.0001）and CD8⁺T_CM（P<0.0001）.3.The WB results showed that the expression of itga1 in CD8⁺T_EMwas significantly higher than that of CD8⁺T_NA（P<0.0001）and CD8⁺T_CM（P<0.05）.4.The results of ELISA showed that the expression of CCL3 in CD8⁺T_EM was significantly higher than that of CD8⁺T_NA（P<0.0001）and CD8⁺T_CM（P<0.0001）.The expression of CCL4 in CD8⁺T_EM was significantly higher than that of CD8⁺T_NA（P<0.0001）and CD8⁺T_CM（P<0.0001）.The expression of IFN-γin CD8⁺T_EM was significantly higher than that of CD8⁺T_NA（P<0.0001）and CD8⁺T_CM（P<0.0001）.5.The results of CFSE staining and ELISA showed that the itga1 locus of CD8⁺T_EM was blocked by itga1 monoclonal antibody,and the proliferation index of CD8⁺T_EM was significantly decreased（P<0.05）,and the amount of CCL3,CCL4 and IFN-γwas sreted.There was also a significant decrease（P<0.0001）.The itga1 monoclonal antibody exerts a significant inhibitory effect on the proliferation and activity of CD8⁺T_EM.6.The itga1 site of CD8⁺T_EM was blocked with the itga1 monoclonal antibody,and the WB results showed that the expressions of p-PI3K/PI3K,p-Akt/Akt,p-p21/p21,p-p27/p27,CDK6 which on the PI3K-Akt signaling pathway,were significantly decreased（P<0.05）.The PI3K site of CD8⁺T_EMwas blocked by PI3K small molecule blocker.The WB results showed that the expression of p-Akt/Akt,p-p21/p21,p-p27/p27 and CDK6 in PI3K-Akt signaling pathway was significantly decreased（P<0.05）.This result demonstrates that itga1 exerts a regulatory effect on CD8⁺T_EM proliferative activity through the PI3K-Akt signaling pathway.7.In vivo results of an allogeneic mouse re-organ（heart）transplantation model showed that the acute rejection of the CD8⁺T_EM-mediated allogeneic mouse re-organ（heart）transplantation model was treated with the itga1 monoclonal antibody.The survival time of the heart increased from 4.5±1.0 days to 8.8±1.8 days,and the difference was statistically significant（P<0.001）.The histopathological grade of the degree of acute rejection of the transplanted heart decreased from0.833±0.753 to 2.670±0.516,and the difference was statistically significant（P<0.001）.The levels of CCL3,CCL4,and IFN-γin the serum of recipient mice treated with the itga1 monoclonal antibody were signific antly lower than those in the recipient mice treated with the itga1 monoclonal antibody（P<0.001）.Three days after transplantation,the proportion of CD8⁺T_EM in the transplanted hearts of recipient mice treated with the itga1 monoclonal antibody was significantly lower than that of the recipient mice treated with the itga1 monoclonal antibody（P<0.001）.The proportion of CD8⁺T_EM in the spleen of recipient mice treated with the itga1 monoclonal antibody was significantly lower than that in the spleen of recipient mice not treated with the itga1 monoclonal antibody（P<0.0001）.These results demonstrate in vivo that the itga1 monoclonal antibody is effective in inhibiting CD8⁺T_EM-mediated acute rejection of allogeneic mouse re-organ（heart）transplantation.Conclusion:1.Successful application of single-cell sequencing technology to the field of organ transplant rejection.2.Itga1 is highly expressed on CD8⁺T_EM and exerts its regulation on its proliferative activity through the PI3K-Akt signaling pathway.Itga1 is a key molecule affecting the proliferation and activity of CD8⁺T_EM.3.The use of itga1 key molecule antagonist itga1 monoclonal antibody can significantly inhibit the proliferation and activity of CD8⁺T_EM cells,and effectively alleviate the acute rejection of allogeneic mouse re-organ（heart）transplantation induced by CD8⁺T_EM,and prolong the survival of transplanted hearts.4.The conclusion of this study provides a new idea for clinical research and development to effectively inhibit the new treatment of acute rejection caused by immune memory.