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 recently adapted the CAR (chimeric antigen receptor) approach to create antigen-specific T-cell populations (T effectors and regulatory T cells/Tregs) from PBMC of normal donors by retroviral transduction of T-cell receptors isolated from a hemophilia subject's FVIII-specific T-cell clone.  The transduced Tregs suppressed both effector T-cell proliferation and production of multiple cytokines.  Importantly, these engineered Tregs also blocked the antibody response to FVIII in vitro (Kim et al.  Blood 125: 1107, 2015).  In collaboration with Dr. Christoph Knigs and Anja Naumann, we have also engineered a single chain (scFv) anti-FVIII antibody into expanded PBMC and shown that transduced Tregs specifically suppressed T effector cell proliferation to FVIII.  In this proposal, we wish to extend this approach by utilizing retroviral transduction of cytotoxic T cells with scFv anti-FVIII or FVIII domains to create CAR CD8 T cells capable of killing antigen-binding B cells (both mouse and human) to further suppress the inhibitor response.  Thus, we will combine two approaches to create specific CD8 cytotoxic cells to directly target B cells and plasma cells bearing FVIII- specific receptors.  Single chain antibodies recognizing the immunodominant FVIII domains will be inserted into a retroviral vector, analogous to CAR strategies, with transmembrane and signaling sequences that will allow triggering of cytotoxicity in CD8 T cells upon recognition of FVIII captured by specific B cells.  A second set of CAR CD8 T cells will be transduced with immunodominant FVIII A2 and C2 domains.  Our hypothesis is that B cells recognizing these domains will be targeted to commit suicide when bound to transduced CD8 T cells.  We will test cytotoxicity directly in vitro by culturing the FVIII-transduced human T cells with hemophilia subjects' B cells and with FVIII-specific hybridomas, and in vivo by adoptive transfer of transduced murine T cells into immunized recipients.  Therefore, our specific aims are: (1) to develop retroviral vectors to express scFv anti-FVIII or FVIII domains in human and mouse CD8 T cells and (2) to determine the effect of engineered FVIII-specific CD8 T cells on FVIII-specific B cells and inhibitor formation.  These studies will provide proof of principle that CD8 CAR T cells can be engineered to effectively eliminate inhibitor responses to FVIII.