The Role Of Costimulatory Blockade And Alloreactive CD4~+ Memory T Cells In Survival Of Allografts

Part I Establishment of ventral heterotopic heart transplantation in miceObjective To explore the improved technique of mouse ventral heterotopic heart transplantation and build animal model on the study of transplantation immunity. Methods The donor heart aorta and the recipient ventral aorta, the donor pulmonary artery and the recipient inferior caval vein, were anastomosed by using the end-to-side suture technique respectively. At the same time, we measured the length of some transplantation-related parts.Results The diameter of recipient inferior caval vein and ventral aorta was(2.4±0.4)mm,(1.5±0.2)mm respectively. The average length of ventral blood vessels which could be used was (6.0±1.0)mm. The succeeding rate was 92 %. The average time of donor operation and recipient operation was (8.0±1.0)min,(40.2±3.0)min respectively. The time of artery anastomosis and vena anastomosis was (9.3±1.2)min,(7.8±1.7)min respectively.Conclusions The animal model was stable and could be used for the study of transplantation immunity. Part II The mechanisms of survival prolongation of murine cardiac allografts using the treatment of CTLA-4 Ig and MR1Objective The present study was undertaken to determine the role of costimulatory block therapy in murine cardiac transplant model and collect some evidences accounting for the mechanism of transplantation immunity.Methods In our study, we blocked the CD28/B7 and CD154/CD40 costimulatory pathway by the transient administration of CTLA-4 Ig and MR1 antibody and then proceeded to study that how did the combination administration of CTLA-4 Ig/MR1 influence on survival time of allografts, deviation of Th1 and Th2 cytokines secretion and other mechanisms related to survival prolongation of allografts.Results Costimulatory blockade can prolong the survival time of cardiac allografts (MST: 43 d for treated group vs. 8 d for untreated group, P<0.01). The costimulatory blockade therapy can downregulate the expression of both Th1 cytokine(IFN-γand IL-2)and Th2 cytokine(IL-4 and IL-10), reduce the quantities of graft-infiltrating CD4+ and CD8+ lymphocytes and inhibit the expression of both perforin/GrB and FasL in allografts.Conclusions The combination administration of CTLA-4 Ig/MR1 can inhibit acute rejection reaction in murine cardiac allografts and prolong the survival time of cardiac grafts through several mechanisms including inhibition of the Th1 and Th2 cytokines expression, graft-infiltration of CD4+ and CD8+ T lymphocytes and both perforin/GrB and FasL-Fas mediated cytotoxicity.Part III The role of alloreactive memory T cells in survival of allograftsObjective To study the role of alloreactive memory T cells in survival of allografts. Methods To draw assistance from mouse skin transplantation model and separate the splenocytes of recipients. The CD4+ Tm were isolated by magnetic activated cell sorting(MACS). The activity and the purity of the isolated cells were detected. And then we transferred these cells into recipients, explored that how did these cells influence the survival of allografts.Results The activity of the CD4+ memory T cells was (98.4±0.7)%. The proportion of CD4+CD44+CD62L-CCR7- lymphocytes were 95% approximately and its function was donor specific. When CFSE labelled Tm were transferred into recipents via tail vein injection 1 day before operation, the fluorescence intensity of these cells fell off gradually. The mean survival time of allografts in Tm group was shorter compared with negative group(no transferred Tm). There were more CD4+ lymphocytes infiltrating into the middle layer of allografts in Tm group compared with negative group. The level of granzyme B in allografts was very low and there were lots of graft-infiltrating FasL+ lymphocytes in Tm group, especially those locations near epicardium, endocardium and around the vessels.Conclusions We could use Magnetic activated cell sorting isolation kit to isolate CD4+ memory T lymphocytes aiming at skin allograft effectively, which facilitates us to investigate the role of memory T cells in transplantation immunity. These alloreactive CD4+ Tm could mediate reject reaction and shorten the survival time of allografts. The main mechanisms of allografts rejection maybe contain FasL-Fas pathway mediated cell apoptosis.