Type 1 diabetes (T1D), autoimmune pediatric disease, occurs due to hyperactivation of self-reactive T lymphocytes, which destroy pancreatic β cells. Suppression by regulatory T cells (Tregs) is a key mechanism limiting such autoreactivity. T1D patients have quantitative and functional Tregs defects. New clinical trials, transferring autologous ex vivo-expanded Tregs into T1D patients, showed signs of success. Our proposal targets major improvement of the current Treg therapies. We aim to create and validate a novel, antigen-specific cellular therapy for T1D. Hence, we designed β cell-specific Chimeric Antigen Receptors (CARs), to be optimized and tested herein. β cell-specificity is conferred by the scFv fragment of an antibody recognizing unique human β cell marker NTPDase3. We hypothesize that engagement of β cellspecific CAR(s) on T cells will induce proper CAR signaling and cause activation and expansion of CAR T cells. When such signaling occurs in CAR-bearing Tregs, additionally modified for sustained immunosuppression through augmented expression of the essential for Tregs function transcription factors Foxp3 and Eos, it will enable local long-lasting antigen-dependent silencing of diabetogenic autoimmunity. This hypothesis will be tested in the following Specific Aims: Aim 1. Design and produce β cell-specific CAR(s), optimize their binding strength, and interrogate their signaling potential in vitro. As either too strong or weak binding of our CARs to β cell antigen will likely provide inadequate activation, we will model our paratope in silico to design mutated variants with optimal antigen binding. Optimized mutant scFv-CAR constructs will be analyzed and CARs that provide constitutive antigen-specific signaling without subsequent T cells exhaustion will be prioritized. Aim 2. Develop and test strategies allowing for maintenance of continued stable and robust suppressive phenotype of β cell-specific CAR Tregs. We will test our inventive strategy creating autologous CAR-bearing induced T regs (iTregs) from the bulk of conventional CD4+ T cells. For that CAR cassettes are designed to provide constitutive ectopic expression of crucial for Tregs development and maintenance transcription factors Foxp3 and/or Eos. CAR iTregs will be tested functionally for their suppressive abilities against human β cells-reactive diabetogenic T cell clones. Completion of our project will confirm the feasibility of β cell-specific CAR Tregs, evaluate their potency, and pave a road for future pre-clinical and clinical trials of CAR Tregs in T1D.