Establishment Of In Vivo Donor Specific Cytotoxicity Assay And Its Application In Transplant Rejection Monitoring

BackgroundAfter organ transplantation an immunosuppressive regimen is required to prevent graft rejection. Immunosuppressive drugs inhibit immune function by targeting both T- and B-cell responses through blockage of cellular proliferation induced by alloantigen stimulation, and by inhibition of the cytokine production necessary for such stimulation. However, the absence of discrimination between the immune response against alloantigen from the transplanted organ and the immune response against environmental antigens renders transplanted patients strongly immunodeficient and susceptible to bacterial and viral infection. Optimising the immunosuppressive drug regimen to balance mandatory immunosuppression and preserving immunity is a difficult challenge for clinicians in charge of transplanted patients. Preserving immunity by minimizing immunosuppression or inducing tolerance is one of the major goals of the transplant immunologist. Several strategies are exciting, but further work is necessary to find the best protocol to induce tolerance. Defining the ideal strategies for inducing tolerance or minimizing the role of immunosuppressive drugs, and development of assays to measure tolerance and immunity, are among the most important challenges in organ transplantation over the next few years.A reliable index of immune status could result in customization of immunosuppressive drugs, not only in the context of rejection/tolerance, but also in the context of a strong increase in susceptibility to infections. Such an objective is highly desirable, given the morbidity and mortality associated with long-term administration of immunosuppressive therapy. However, although monitoring of immune function (with the use of analysis of blood or graft specimens) has helped delineate the host's response to the graft (including the phenotype and function of infiltrating cells, alterations in the T-cell repertoire, and the types of cytokines and antibodies produced), no test has yet proved to be a reliable method in detecting rejection. Therefore, the combination of clinical assessment and a graft biopsy is still a useful and prevalent pattern to determine the immune status of the recipent. However, the biopsy is a harmful approach for the graft and the host.In this paper we describe a simple method using the vital dye 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) to follow donor splenocytes rejection by flow cytometry, and recipients splenocytes as control in vivo. Labelled spleen cell mixtures were injected intravenously into various recipients and blood samples were taken at different time points to follow the transferred cells. We found that the labelled recipient cells could be readily detected by flow cytometry for up to six days, and the loss of these labelled donor cells in various transfusion experiments with different major and minor histocompatibility differences followed the rejection kinetics of skin transplants. Thus, this method offers a simple and effective tool to test the immune status of the host in transplantation experiments in which donor and host are not completely syngeneic. We have also demonstrated that the method is specific, sensitive, precise, and well suited for quantitative immunological studies. ChapterⅠObjective: To establish a method of donor specific cytotoxicity cytotoxicity assay in vivo for detecting the immune status of the recipient using flow cytometry.Methods:C57BL/6j (H-2b)and Balb/c(H-2d) splenocytes were harvested, labelled by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) at a final concentration of 0.3 and 6 uM, respectively.A skin transplantation model was established with the recipients BALB/c mice and the donors C57BL/6 mice. Donor, inbred C57BL (C57; H-2b) mice, female, 6-8 wks old; Recipient, inbred Balb/c (BALB; H-2d) mice, female, 6-8 wks old. The transplantation models were divided into 8 groups (1h,2h,4h,10h,1d,2d,3d,6d) Similarly, Balb/c mice without skin transplantation served as negative control. After intravenous transfusion of the CFSE-labelled donor/recipient splenocytes mixture, the following examinations are carried out at different time points.The total number of CFSE-positive cells was subsequently determined in PBMC at eight different time points following adoptive transfer (from 60 s to 1 week), providing a quantitative estimate of the ratio of donor/recipient splenocytes labelled with CFSE in host peripheral blood.The percent specific lysis was calculated using the equation:% specific lysis=1—donor cells/recipient cells×100%Results:1 The average survival times of the allografts in Balb/c mice of the model groups were 13days.2 In Balb/c mouse rejection model, we demonstrate how to obtain a direct, unbiased estimate of the total number of adoptively transferred cells in PBMC at different time points. The estimate is obtained by a straightforward method based on flow cytometry. The total number of CFSE-positive cells was subsequently determined in PBMC at eight different time points following adoptive transfer (from 60 s to 1 week) in model and control groups.3 Compared with the control groups, the percent specific lysis of donor cells in the rejection model mice lh, 2h, 4h,10h, ld,2d, after transfer were all significantly higher than those of normal Balb/c mice t=39.5, 41.1, 26.2, 27.4, 15.Sand 5.5, all P＜0.01). In the control groups, the percent of specific lysis rapidly increased 87.4%±4.5%. at 1h after transfer, and at 2h the percent was 92.2%±3.9%.Conclusion:We demonstrate that the method is simple, precise, reliable and well suited for quantitatively detecting the immune status of skin transplant recipients in vivo. Consecutive surveillance of the percent of specific lysis is helpful in the early detection of acute rejections, and in judgment of the therapeutic effect of immunosuppressive drugs and possibility of infection.ChapterⅡTo study specificity of the cytotoxicity assay in vivo.Objective: To investigate specific lysis of donor cells in peripheral blood in skin transplant recipients by cytotoxicity assay in vivo.Methods:1 CFSE-labelled donor/recipient splenocytes mixture and third party DBA mice/recipient splenocytes mixture were prepared;2 The cytotoxicity assay in vivo were performed to analysis specific lysis of donor cells and third party splenocytes in peripheral blood in skin transplantation Balb/c mice models3 The ratio of CFSE-positive donor/recipient cells and third party DBA mice/recipient splenocytes was subsequently determined in PBMC by flow cytometry at eight different time points following adoptive transfer (from 60 s to 1 week) in different groups.Results:the percent specific lysis of donor cells in the rejection model mice 2h, 4h after transfer were all significantly higher than those of third party DBA mice splenocytes (Post hoc multiple comparisons by S-N-K).Conclusion: The assay is confirmed as an specific and rapid method of quantitatively detecting the immune status of skin transplant recipientsChapterⅢTo study sensibility of the cytotoxicity assay in vivo and its application in transplant rejection mornitoringObjective: 1 To investigate whether the cytotoxicity assay in vivo is a sensitive method for detecting more subtle changes in the immune status and determine whether this approach can detect sub-clinical rejection.2 To expand the range of application of the cytotoxicity assay in vivoMethods:1 A skin transplantation model was established with the recipients BALB/c mice and the donors C57BL/6 mice. the recipients BALB/c mice were divided into 13 groups according to the days of after skin transplantation (1d-13d, n=5). The immune status of the every group recipients were detected by the cytotoxicity assay in vivo.2 Models of liver transplantation were established with the recipients SD rats and the donors Wister rats, and long-term living models (No less than 1 month) were induced by donor apoptosis cells, the immune status of the long-term living models were detected by the cytotoxicity assay in vivoResults:1 These tests, performed serially after transplantation, are sensitive enough to detect sub-clinical rejection 2 Compared with the control groups, the percent specific lysis of donor cells in liver transplantation model 2h after transfer were significantly lower than those of acute rejections rat by skin transplantation (t=10.6, P＜0.001).Conclusion:1 The cytotoxicity assay in vivo is a sensitive method for detecting the immune status of the skin transplantation recipient.2 The cytotoxicity assay in vivo can apply to monitor the immune status of liver transplantation rat.