A major problem in the treatment of hemophilia A patients with factor VIIII (FVIII) is that up to 30% of these patients produce antibodies to therapeutic FVIII. These antibodies block (inhibit) the pro- coagulant function of FVIII and thus are termed inhibitors. The focus of our lab has been to develop novel approaches for the induction of tolerance so that it can be applied to the prevention or reversal of undesirable immune responses, including the formation of hemophilia inhibitors. We have used immunoglobulin (Ig) fusion proteins expressed in B lymphocytes for the induction of tolerance in animal models of hemophilia. This technology is based on the well-known tolerogenicity of IgG carriers, onto which we engineer multiple epitope-containing polypeptides in frame at the N-terminus. The multiple epitopes processed and presented on MHC class II lead to tolerance and clinical efficacy in multiple autoimmune models and in naive and even primed hemophilia A mice. We know that CD4+CD25+ regulatory T cells (Tregs) are required in this process and have shown that that the IgG scaffold increases the efficacy of this tolerance process. Independent data suggests that IgG domains contain epitopes, termed Tregitopes, which activate Tregs. We hypothesize that the presence of Tregitopes and IgG processing mechanisms promote tolerogenic presentation. In this revised renewal, we propose to further define the mechanisms of tolerogenic processing in B cells and to directly target B cells using the CD20 marker. T cell receptors from hemophilia patients (and their clones per se) will be used to move these studies forward for translation to the clinic. Our Specific Aims, thus, are: (1) to determine the role of regulatory T cell epitopes and IgG processing in tolerance, (2) to use B-cell targeted vectors to modulate responsiveness, and finally (3) to use retrogenic mice expression human TCR specific for FVIII and transduction of expanded human regulatory T cells to understand the mechanism of tolerance and to effectively suppress responses to FVIII. Elucidating these pathways will enhance our ability to optimize the efficacy and safety of this approach for use in humans.