The goal of this proposal is to facilitate the development of monoclonal (mAb) anti-CD154 therapy in transplant rejection by elucidating the mechanism by which anti-CD154 enhances graft acceptance. Central to this project are unique reagents and systems that we have developed to study CD154 biology. A critical element of CD154 biology is its transience of expression which is thought to restrict helper/inducer function to appropriate CD40+ cell targets and limit triggering of inappropriate (and potentially autoreactive) targets. The transience of CD154 expression is mediated in part by CD154 endocytosis which may also serve to disengage helper cells from targets and render helper cells refractory to providing helper signals to other targets. Therefore, we propose to analyze the role of the cytoplasmic domain of CD154 in the process of CD154 endocytosis and to characterize the molecular interactions that drive this process. The cytoplasmic tail of CD154 will be characterized by mutagenesis, phosphorylation analysis, and for its binding to downstream signaling proteins and components of the endocytic pathway. Since CD154 may also signal """"""""backwards"""""""" to the T cell, the action of anti-CD154 antibodies on transplant acceptance may reflect distinct effects on CD154 """"""""backwards"""""""" signaling (into the T cell) and on CD40 """"""""forward"""""""" signaling (into the APC). We will directly study the cells of anti-CD154 treated and control animals after organ transplantation, to ascertain whether deficient endocytosis or aberrant CD40 signaling correlates with rejection or acceptance of grafts in these animals. Together, these studies should allow a reductionist approach to understanding the role of the CD154 in immune physiology and transplant rejection. Better understanding of the roles of """"""""backwards"""""""" signaling will facilitate the analysis of therapeutic regimens based on anti-CD154 on transplant acceptance.
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