Abstract Regulatory T cells (Tregs) express FOXP3, a transcription factor encoded on the X-chromosome. Tregs are critical to prevent autoimmunity and maintain immune homeostasis and tolerance. Naïve CD4+ T cells can be converted into “induced” Tregs (iTregs) in culture, but compared to endogenously generated Tregs, iTregs rapidly lose expression of Foxp3 upon cell division or after adoptive transfer. The stability of Foxp3 expression has been linked to the DNA methylation status of an intronic enhancer, Foxp3 CNS2: endogenously generated Tregs are almost fully unmethylated at CNS2, whereas naïve CD4+ T cells and iTregs activated in vitro with TGFβ plus retinoic acid (RA) are almost fully methylated. We discovered several years ago that TET-family dioxygenases alter DNA modification status by oxidizing 5- methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). 5hmC can be further oxidized by TET proteins to 5- formylcytosine (5fC) and 5-carboxylcytosine (5caC). All three oxidized methylcytosines interfere with the main- tenance function of DNA methyltransferase 1, thus effecting “passive” replication-dependent DNA demethy- lation during the course of cell division. Additionally, the DNA repair enzyme thymine DNA glycosylase (TDG) can excise 5fC and 5caC, which are then replaced with unmodified C through base excision repair. We have recently shown that TET enzymes promote CNS2 demethylation in Tregs. By activating naïve T cells in the presence of TGFβ, RA and the TET activator Vitamin C, we can generate exceptionally stable human and mouse iTregs, in which the stability of Foxp3 expression appears equivalent to that of endogenous Tregs. Vitamin C promotes TET-mediated CNS2 demethylation, whereas RA increases Foxp3 stability without CNS2 demethylation. Our goal in this application is to investigate the role of TET methylcytosine oxidases in T cell lineage specification at a molecular and kinetic level, with a focus on iTreg differentiation. Using state-of-the-art technologies and essential reagents developed in our laboratory, we will explore the role of TET proteins at early and later stages of iTreg differentiation (Aim 1); investigate the importance of TDG in DNA demethylation during iTreg cell differentiation (Aim 2); and define the roles of RA and Vitamin C in maintaining Foxp3 stability in dividing iTregs (Aim 3). Our studies have strong clinical relevance, since iTreg cells generated in culture have the potential to be useful in transplant medicine and to cure autoimmune disease. Our proposed studies will enhance our understanding of the very fundamental question of how DNA modification regulates gene transcription, particularly with respect to the stability of expression of lineage- determining transcription factors, which in turn determines the plasticity of cellular lineages. Potentially, our data will also help identify novel candidates for therapeutic intervention in immune-related disorders, including cancer immunotherapy, transplant r...