Suppression Of CD8+ T Cell Mediated Rejection By CD4+CD25+ T Cells

Transplantation has been developed to an effctive strategy for treatment of end stage diseases of multiple organs since 1950's.Despite success of current immunosuppressive drugs, transplantation immunologists and surgeons are still seeking to achieve tolerance to allograft.There are several different mechanisms such as clonal deletion,anergy,neglect,immuno regulation .which maintain tolerance to self-antigen.Some strategies , derived from these mechanisms has been proved to be encouraged in suppression of allograft rejection. Immunoregulation mediated by regulatory T cell is one of the most important mechanisms to maintain self tolerance. Recently, accumulating evidences have shown that regulatory T cells play a key role both in maintenance of self tolerance and tolerance to allograft. In terms of regulatory T cell,CD4+CD25+ T cell is representitive.ln naive individuals,CD4+CD25+T cells function as self tolerance, inrecipients with long term survival allograft.they play an important role in suppression of allograft rejection.Immune regulation potent CD4+CD25+ T cells can be recovered from recipients with long term survival allografts.Such regulatory T cells can suppress T cell mediated rejection in vitro and in vivo. What are the effects of such regulatory T cells on alloreactive CD4+ and/or CD8+ T cells? Is this immunosuppression alloantigen specific? This study focused on suppression of alloreactive CD8+ T cell mediated rejection by CD4+CD25+ T cells. ObjectiveThe aim of this article was to induce long term survival allografts by blocking CD40-CD40L pathway with homemade anti-CD40L monoclonal antibody MR1 ,then purify CD4+CD25+ regulatory T cells from recipients with long term survival allografts, and adoptively cotransfer such cells together with TCR transgenic allogenic antigen specific CD8+ T cells into immunodeffient mouse.finally study whether CD4+CD25+ T cells can suppress allogenic antigen specific CD8+ T cell mediated rejection ,thus clarified whether CD4+CD25+ T cell mediated regulation is one of mechanisms of prolongation ofallograft survival by CD40--CD40L pathway blockade.Methods1 MR1 hybridoma was cultured in hollow fibre system.culture supernatant was harvested,then precipitated by half-saturated ammonia sulphate.MFM was purified by DEAE-sephacel ion exchange chromatography.MRI concentration was measured by ELISA.2 Purified MR1 was used in following experiment.(1) C57BL/10 hearts were heterotopically transplanted into abdomen cavity of CBA mice.500 u g MR1 was injected peritoneally at dayO,2,4 post transplantation.A group of CBA recipients were injected with saline as control.Palpation of transplanted heart beating was performed everyday.The rejection of cardiac graft was determined as stop of beating. Allografts were harvested at different time points for histology and immunhistochemistry.(2) C57BL710 full thick tail skin was cut into 1 1 cm2 pieces.one piece of skin was transplanted onto left flank of CBA mouse.MRI was given the same as in heart transplantation .A group of CBA recipients were injected with saline instead of MR1 as control.In addition, a group of recipients also were injected intravenously 1 107 spleen cells (donor specific transfusion,DST) at the day of transplantation.The survival of allografts was monitored every two or three days.Allografts were harvested at 10 days followingtransplantation.3 Spleens and mesenteric lymph nodes were harvested from CBA recipients with long term survival allocardiac grafts,CD4+CD25+ T cells were purified by MACS negative and positive enrichment.Purity of CD4+CD25+ T cells was estimated by FACS.4 5X105purified CD4+CD25+ T cells were adoptively cotransfered with 1 X105 BM3 RAG TCR transgenic CD8+ T cells into CBA RAG-/- mouse,C57BL/10 tail skin was transplanted onto CBA RAG-/- mouse one day later.A group of mice were transferred only with 1 X105BM3 RAG TCR transgenic CD8+ T cells as rejection control.a group of mice didn't received...