Abstract We propose to develop nontoxic, tunable off-the-shelf chimeric antigen receptor (CAR) cell-based immunotherapy to treat acute myeloid leukemia (AML). Murine CD19 CAR CD4 Treg adoptive transfer fully prevented graft-versus-host disease (GVHD) and eliminated all CD19 lymphoma cells. Surprisingly, the mechanism was not CD4 Treg mediated suppression of Teffectors to below that needed for GVHD but above that needed for graft-vs-leukemia (GVL). Instead, these Tregs had dual suppressor and cytolytic functions as evidenced by equal killing comparable to Teffectors at a Treg purity level of 99.9%. In vitro and in vivo killing was ligand dependent. These encouraging data led to testing of human CD19 CAR CD4 Tregs that also killed in a ligand specific manner. Peripheral blood CAR CD8 induced Tregs (iTregs) were equally potent for target cell killing as CAR CD8 Teffectors. Human CD19 CAR CD4 Treg adoptive transfer alone rescued 50% of mice from a lethal lymphoma dose. With encouraging results, we pursued human pluripotent stem cells (iPSCs) that produce CD8 but not CD4 T cells and are highly amenable to multiplex gene editing due to their self-renewing properties. These data lead to Our central hypothesis that gene-edited off-the-shelf CD83 CAR iPSC CD8 iTregs will avoid rejection and exhaustion but retain dual GVHD/GVL function. Edits are designed for site specific integration or disruption, including Foxp3 (completed), circumventing rejection by macrophages (CD47 over- expression) and the adaptive immune system by deleting HLA class Ia/II, and STAT3 to render Tregs resistant to Teffector conversion. Embedded in genome editing are key biological questions as to how changes in proteins affect the initiation, maturation and function of CD8 iTtregs, providing a great opportunity to study human T cell development and control mechanisms. CAR therapy for AML is challenging due to on-target/off-tumor toxicity. We nominated CD83 as differentially expressed on malignant compared to nonmalignant myeloid cells. A nanobody CAR generated from a camelid library has exquisite potency against AML cells. The requirements for in vivo persistence and function of CAR CD8 iTregs that correlate with efficacy will be addressed here. While persisting CAR T cells may control AML, T cells that undergo tonic or repetitive signaling can become exhausted and unable to control disease. CAR T cells must be tuned for a productive immune response but not overly vigorous to drive cells into a state of dysfunction manifested by poor effector function, inhibitory receptor expression and altered transcriptomics and epigenetics. Strategies ranging from TCRα deletion and reliance on CAR signaling to immunotyrosine activation motif deletion to dampen CAR signaling but leave TCR signaling intact and to switch CARs turned on/off by stopping a drug dimerizer. Aim 1 hypothesizes that genome modified off-the-shelf human iPSCs will generate CD83 CAR CD8 iTregs that resist rejection, augment GV...