Abstract DNA cytosine methylation (hereafter, DNA methylation) has a critical role in cell lineage specification as well as suppression of repetitive and transposable elements in the genome. DNA methyltransferases attach a methyl group to generate 5-methylcytosine (5mC); TET methylcytosine dioxygenases cause DNA demethylation by oxidizing the methyl group of 5mC to 5-hydroxymethylcytosine (5hmC) and beyond. We have shown that TET- deficient cell types display not only the expected increase in DNA methylation at promoters and enhancers, but also a paradoxical decrease in DNA methylation in heterochromatic regions of the genome. The consequences of these molecular features remain to be understood, but similar alterations in genome-wide DNA methylation patterns have been observed in cancer and aging. By studying the phenotypes of several mouse strains in which Cre recombinase was expressed either inducibly or developmentally in immune/ hematopoietic cell types, we showed that deletion of two or more Tet genes skewed cell lineage commitment in the relevant cell type, in a manner that correlated with changes in cell lineage commitment. More striking phenotypes, however, were that Tet2/3 fl/fl CD4Cre mice displayed massive TCR- dependent expansion of iNKT cells; and that Tet2/3 fl/fl Foxp3Cre mice developed a dominant proinflammatory phenotype observed in heterozygous female mice, in mixed bone marrow chimaeras, and in immunocompetent recipients injected with total CD4+ T cells from Tet2/3 fl/fl Foxp3Cre mice. This phenotype differs markedly from that observed in heterozygous Foxp3+/- females and in immunocompetent mice injected with Foxp3-deficient cells, which do not develop disease. In Aim 1, we will address the mechanisms underlying the striking expansion of Tet2/3-deficient iNKT cells by using adoptive transfer approaches in vivo and recently-developed cell culture systems that recapitulate the expansion in vitro. In Aim 2, we will ask whether the dominant autoimmune/ inflammatory phenotype of Tet2/3-deficient T regulatory cells requires, directly or indirectly, the decreased DNA methylation in heterochromatin observed in every TET-deficient cell type examined so far. Decreased DNA methylation in heterochromatin results in “heterochromatin dysfunction”, an aberrant cellular condition linked to autoimmune/ inflammatory disorders, cancer, aging, and neurodegenerative diseases, that stems from aberrant expression of transposable elements (TEs) and resulting DNA damage. DNA damage provokes “sterile inflammation”: activation of innate immune sensing pathways for RNA and DNA with consequent upregulation of type I interferons, interferon-induced genes and proinflammatory cytokines (e.g. IL-1b, IL-6, IFNg, IL-17) Our proposed experiments will add to our knowledge of how TET proteins influence T cell expansion and T regulatory function. More broadly, they will enhance our general understanding of the links connecting TET deficiency and TE expression with a...