PROJECT SUMMARY Type 1 diabetes mellitus (T1D) is an incurable autoimmune disease that results in the destruction of insulin producing β cells and affects nearly 10% of the global population. T1D most commonly affects young children and adolescents and can lead to irreparable and deadly complications if not properly treated. Current treatments and management strategies require time-intensive exogenous insulin administration, with incredible onus on patients and their caregivers. Islet transplantation to replace β cells is an attractive and FDA-approved alternative to insulin therapy in T1D. However, the widespread acceptance of this approach faces formidable challenges, including the absence of safe, non-toxic methods to shield transplanted allogeneic donor islets from immune rejection and a scarcity of donor material. To surmount these hurdles, the field must accomplish two critical objectives: 1) establish an effective, non-toxic strategy to induce tolerance toward donor tissues and 2) identify a readily obtainable source of donor material for transplantation. An emerging field for transplant tolerance is mixed chimerism achieved by hematopoietic cell transplant (HCT), establishing tolerance toward donor-matched tissues and correction of defects that cause autoimmunity. Unfortunately, this requires high doses of radiation and/or chemotherapy to prepare and condition the host prior to HCT, resulting in undesirable toxicities. The development of low intensity conditioning protocols with reduced toxicity, adverse effects, and risk for graft vs. host disease (GVHD) is necessary to increase access to HCT for patients without imminent fatal illness or malignancy. Additionally, pluripotent stem cell (PSC)-derived β-like cells offer an attractive and unlimited source of material for islet cell replacement therapy. However, the immunogenicity of PSC derivatives and the potential for mixed hematopoietic chimerism to induce tolerance to these derivatives remains unexplored. In recent exciting work, we show that a novel low intensity conditioning protocol using monoclonal antibodies for T cell and hematopoietic stem cell (HSC) depletion combined with a non-myeloablative dose of total body irradiation allows for stable mixed chimerism and donor-matched allogeneic islet transplant tolerance. Here, we propose to 1) optimize our conditioning protocol to significantly reduce, or eliminate, the radiation dose required for conditioning and generation of mixed hematopoietic chimerism across full MHC barriers, and 2) evaluate the immunogenicity of mouse PSC-derived β-like cells and tolerance induction in mixed hematopoietic chimeras. Collectively, this research will develop reduced intensity conditioning regimens to produce sustained mixed chimerism and islet transplant tolerance. This will allow for use of this technique beyond patients with fatal malignancy or autoimmunity, including in patients with T1D, as well as provide the necessary training for me to be...