Study On The Role And Mechanism Of Memory CD8 ~ + T Cells In Acute Rejection Of Allograft

PART ONEESTABLISHMENT OF THEHETEROTOPIC ABDOMINAL HEART TRANSPLANTATION MODEL IN MICEABSTRACTObjective:To establish a heterotopic abdominal heart transplantation model of BALB/c mice. Methods:Sixty C57BL/6J mice and sixty BALB/c mice were used as heart donors and receptors, respectively, in the heterotopic heart transplantation. Donor hearts were transplanted into the abdomen of recipient mice. The ascending aorta and pulmonary artery of donor hearts were connected with the abdominal aorta and inferior vena of recipient mice, respectively, by end-to-side anastomosis under microscope. Results:Among the60cases of heterotopic heart transplantation,49cases succeed and11cases failed, with a success rate of81.7%. Reasons of transplantation failure included excessive bleeding, poor venous return, lower limb paralysis, and deep anesthesia. Conclusion:We successfully established a mouse model of heterotopic abdominal heart transplantation. The transplantation surgery requires skilled techniques and the high success rate of heterotopic abdominal heart transplantation in mice can be achieved by systemic practices. Our heterotopic abdominal heart transplantation model in mice provides a useful platform for the investigation of immune tolerance of organ transplantation. PART TWOINDUCTION, PURIFYICATION AND IDENTIFICATION OF MEMORY CD8+TCELLS IN MICEABSTRACTObjective:To induce, isolate, and purify memory CD8+T cells in BALB/c mice, to identify their distributions in mice. Methods:Sixty BALB/c mice were randomly divided into two groups:the experimental group and the control group. BALB/c mice in the experimental group received skin grafts on the back from C57BL/6J mice, but the control mice didn’t receive skin grafts. Three months after the skin graft, memory CD8+T cells and non-sensitized CD8+T cells were isolated and purified using magnetic bead-based isolation technology and the surface markers of CD8+T cells were analyzed by flow cytometry. Memory CD8+T cells stained with DiR were injected into mice via the tail vein and the distribution of memory CD8+T cells in mice and dynamic changes of fluorescence signal were examined by in vivo imaging technology. Results:We successfully isolated and purified memory CD8+T cells and non-sensitized CD8+T cells from the mouse spleen using magnetic bead-based isolation technology, with a purity of96.4% and96.3%, respectively. We found that majority of memory CD8+ T cells accumulated in the liver and spleen after they were injected into mice via the tail vein and the fluorescence signal was undetectable after72hours of tail vein injection. Conclusion:The magnetic bead-based isolation technology can be used for the isolation and purification of memory CD8+ T cells and non-sensitized CD8+ T cells from the mouse spleen with high purify. The memory CD8+ T cells mainly distributed in the liver and spleen after being injected into mice. PART THREETHE ROLE AND MECHANISM OFMEMORY CD8+T CELLS IN THE ACUTE REJECTION OF HETEROTOPICABDOMINAL HEARTTRANSPLANTATION IN MICEABSTRACTObjective:To investigate the role and mechanism of memory CD8+T cells in the acute rejection of heterotopic abdominal heart transplantation in BALB/c mice. Methods:Thirty-six BALB/c mice were randomly divided into three groups (n=12in each group):the experimental group received sensitized CD8+ T cells and cyclosporine A, the control group1received naive CD8+ T cells and cyclosporine A, and the control group2received cyclosporine A without infusion of any T cells. C57BL/6J mice were used as heart donors. Heart transplantation was conducted after three weeks of infusion of CD8+ T cells into recipient BALB/c mice. Intraperitoneal injection of cyclosporine A (5mg/kg/d) was performed from one day before the heart transplantation until graft arrest. The allograft survival time and histological grade of acute heart transplant rejection were compared among mice of the three groups. The percentage of memory CD8+T cells in the spleen was determined by flow cytometry and compared among three groups. The serum levels of IFN-y and CXCL-9in mice of three groups were measured by ELISA and RT-PCR was performed to evaluate the levels of TGF-β,IFN-γ, and IL-2in cardiac allografts. Results:The survival time of heterotopic cardiac allografts in the experimental group (receiving CD8+T cells and cyclosporine A) was significantly shorter than the two control groups (P <0.05). In the control group1, the survival time of heterotopic cardiac allografts was shorter than that in control2group, but the difference was not statistically different (P>0.05). The histological grade of acute heart transplant rejection in the experimental group was significantly higher than that in the two control groups (P<0.05), but no significant difference was observed between the two control groups (P>0.05). Three days after the heart transplantation surgery, the percentage of memory CD8+T cells in the spleen of the experimental group mice was significantly lower than that in the two control groups (P<0.05). In addition, the serum levels of IFN-y and CXCL-9in the experimental group mice were significantly higher than that in the two control groups (P＜0.05), but no significant difference was observed between the two control groups (P>0.05). The levels of TGF-β in cardiac allografts was significantly lower (P<0.05), but the levels of IFN-y and IL-2in cardiac allografts in the experimental group mice when compared to the two control groups (P<0.05). Conclusion:Re-exposure of cardiac allograft to memory CD8+ T cells caused acute rejection that was not attenuated by the application of cyclosporin A. Re-contact between memory CD8+T cells and alloantigen can increase the level of chemokine CXCL-9that attracts memory CD8+T cells to accumulate in the inflammatory sites to cause acute rejection via the secretion of cytokines IL-2and IFN-y.