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In Vivo Donor Specific Cytotoxicity Assay and Its Application in Rabbits Receiving Skin Transplants

Author: ZhongLiMin
Tutor: PanMingXin
School: Southern Medical University,
Course: Hepatobiliary Surgery
Keywords: CFSE Flow cytometry Cytotoxicity assay Graft rejection Celltrafficking Immune monitoring
CLC: R392
Type: Master's thesis
Year: 2012
Downloads: 29
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Abstract


BackgroundOrgan transplantation is an effective treatment of end-stage diseases, cancer and many other diseases, After 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 dye5-(and6-) 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, 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.Objective:To establish a method of donor specific cytotoxicity assay in vivo for detecting the immune status of the rabbits receiving skin transplants using flow cytometry. Understand the antigen-specific of donor specific cytotoxicity assay in vivo. Transplant rejection model is theory of antigen-specific, only reject donor compound antigen. Our results further confirm the specificity of this method.Methods:(female) rabbits and recipient (male) rabbits splenocytes were harvested, labelled by5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) at a final concentration of0.24and6uM, respectively.A skin transplantation model was established with the recipients(male) rabbits and the donors (female) rabbits. The transplantation models were divided into5groups (allogeneic (A), autogenic (B), control (C), and low-(D) and high-dose (E) immunosuppression groups). 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 four different time points following adoptive transfer (from1hour to8hours), 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:In the allograft rejection group, the graft became to turn black by the ninth day post-transplantation. Skin graft rejection was defined as complete skin necrosis. Mean graft survival was13.0±1.0day. In the low-dose immunosuppression group, transplant rejection occurred on the twelfth day post-transplant and mean survival time was13.4±1.1days. In the autograft group, mean graft survival was>50days, and mean survival time of the skin grafts in the high-dose immunosuppression group was23.2±1.5days.Histologically, necrosis of the skin graft, damaged skin structure, and massive lymphocytic infiltration were observed in the allograft rejection and low-dose immunosuppression groups. In the autograft and high-dose immunosuppression groups, the skin grafts had no histologic changes indicative of necrosis:the skin structure was normal, without lymphocytic infiltration.The kill rates of the allograft rejection and low-dose immunosuppression groups reached their highest levels (86.19±6.95%and88.14±4.21%, respectively)8hr following injection of the spleen cell suspension. Allogeneic spleen cells from donor male rabbits were almost completely removed within8hr of injection. The kill rate increased slowly in the nontransplant (control), autograft, and high-dose immunosuppression groups without specific killing.Conclusion:We demonstrate that the method is simple, precise, reliable and well suited for quantitatively detecting the immune status of skin transplant rabbit 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.

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