Akt-induced endothelial activation in alloimmune responses

Recent findings demonstrated that the status of the graft itself plays a dominant role in shaping the outcome of allograft rejection by controlling the trafficking of effector T cells. In the context of transplantation, the endothelium is unique, as it is the primary interface between the recipient blood and the donor tissue. Pro- inflammatory cytokines can mediate endothelial activation, so it is proposed that the nature of the T cell activation response ultimately regulates graft endothelium mediated events. Thus it is difficult to ascertain how selective events mediated by the endothelium alone are able to regulate allograft rejection and to affect alloimmune tolerance. Many cytokines induced in allografts during rejection are established to activate the PI3-Kinase-Akt signaling pathway in endothelial cells. In this project a unique transgenic mouse model allows to address key questions related to the selective role of Akt-induced donor endothelial cell activation in alloimmune responses and in alloimmune tolerance. These are important questions, as selective suppression of endothelial cell activation responses might prevent allograft rejection and maintain alloimmune tolerance. First the expression of the activated form of the kinase Akt in endothelial cells in association with allograft rejection will be analysed. A double transgenic mouse (VE-cadherin;tTA/TET-Akt) will then be used - in which it is possible to selectively regulate endothelial activation via expression of Akt under the control of tetracycline - to determine the function of Akt-induced endothelial cell activation in the regulation of alloimmune T cell responses and to evaluate the effect of donor endothelial activation on alloimmune tolerance in vivo. To achieve these goals the VE-cadherin;tTA/TET-Akt mice (C57BL/6 H-2b ) will be used in transplantation studies as donors of cardiac allografts and BALB/C (H-2d ) mice as recipients. Isolated transgenic endothelial cells will be co-cultured with recipient CD4 + T cells, CD8 + T cells and CD4 + CD25 + regulatory cells to examine whether Akt-activated endothelial cells can mediate interactions with T cells, as a primary event, and facilitate T cell activation and T cell transendothelial migration, and influence the ability of regulatory T cells to suppress T cell activation. In order to assess the role of donor endothelial cells in initiation and maintenance of tolerance in vivo, tolerance in recipients of VE-cadherin;tTA/TET-Akt double transgenic cardiac allografts will be induced using CTLA4Ig/MR1. The response will be evaluated by analysis of allograft survival, allograft histology, inflammatory infiltrates, expression of chemokines and adhesion molecules in allografts, and of effector CD4 + and CD8 + T cells and regulatory T cells. A graft endothelium at a quiescent state might be vital for the maintenance of alloimmune tolerance and for long-term graft survival. Together this project will determine the pathophysiological function of Akt-induced endothelial activation in the regulation of alloimmune T cell responses and in alloimmune tolerance.