ADOPTIVE TRANSFUSION OF EX VIVO DONOR ALLOANTIGEN-STIMULATED CD4+CD25+ REGULATORY T CELLS PREVENTS COMPOSITE TISSUE ALLOTRANSPLANTATION REJECTION

• Main Authors: Sherilyn Keng Lin Tay, 鄭慶玲
• Other Authors: F. C. Wei
• Format: Others
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/27671405884936519916

碩士 === 長庚大學 === 生物醫學研究所 === 98 === Introduction: There is increasing evidence that regulatory T cells (Tregs) play an important role in preventing allograft rejection in both experiment and clinical settings of solid organ transplantation. Adoptive transfusion of alloantigen-stimulated Tregs have been shown to ameliorate rejection of solid organ transplantations, however this has not been shown in composite tissue allotransplantation. Therefore, the therapeutic potential of Tregs were investigated in a rat hindlimb osteomyocutaneous composite tissue allotransplantation. Methodology: The role of Tregs was first quantified in tolerant rats created using an established CTA model by flow cytometry. Using the same model of composite tissue allotransplantation, hindlimbs from Brown-Norway rats were heterotopically transplanted onto Lewis rats based on the femoral vessels. Freshly isolated (n=5) or ex vivo alloantigen stimulated (n=3) CD4+CD25+ Tregs (3x106cells) from naïve Lewis rats were injected into the rats on day 11 after transplantation. Recipient rats were preconditioned with antilymphocyte serum on days -1 and day 2 of the transplant and given daily subcutaneous cyclosporine from day 0 to day 7. Tregs were harvested and isolated from splenocytes of naïve Lewis rats. These were sorted using magnetic beads and AutoMACS system. For ex vivo alloantigen stimulation, isolated Treg cells were co-cultured with irradiated antigen presenting cells from naïve Brown-Norway rats for 72 hours before transfer. The suppressive effect of these cells was investigated using one-way mixed lymphocyte reaction (MLR) and the rats monitored daily for signs of rejection. Histological analyses of the flap were taken at defined points and the Treg profile assessed. Results: Both the level of CD4+CD25+ Tregs and the CD4+CD25+/CD4+CD25- ratio were increased in both splenocytes* and peripheral blood of tolerant rats compared to naïve rats. In MLR, ex-vivo alloantigen stimulated CD4+CD25+ suppressed antigen-specific Lewis splenocyte proliferation in a dose dependant manner and this was significant at a 1:1 ratio. Adoptive transfer of 3x106 of these cells prolonged the survival of the transplanted grafts* and resulted in long term acceptance of the graft in 33% of the rats. This rat shows minimal rejection on histological analysis and similar to tolerant rats show an increase in CD4+CD25+ cells and the CD4+CD25+/ CD4+CD25- ratio. * denotes p<0.05 Conclusion: Adoptive transfusion of ex vivo donor alloantigen-stimulated CD4+CD25+ Tregs combined with a short course of cyclosporine and T cell depletion may represent a clinically feasible strategy for preventing rejection of composite tissue transplantation and tolerance induction.