TET2 and 3 proteins control iNKT cell lineage specification and TCR mediated expansion

Abstract TET proteins are 2-oxoglutarate- and Fe(II) dependent dioxygenases that catalyze the hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products (5-formylcytosine and 5-carboxylcytosine) in DNA. They can act as mediators of “active” (replication-independent) DNA demethylation, achieved through excision of 5fC and 5caC by thymine DNA glycosylase (TDG) followed by replacement with an unmethylated cytosine by base excision repair. They are also novel epigenetic marks that are recognized specifically by readers. The role of TET proteins in developing thymocytes remains elusive. In the present study, we show that simultaneous deletion of TET2 and TET3 in mice (DKO) results in aberrant development of invariant NKT cells that are skewed towards the NKT17 lineage. We also found upregulation of proliferation controlling genes as well as genes that are normally expressed in precursor cells. This results in an unprecedented expansion of iNKT cells, leading to lethality. The expansion is driven by antigen recognition, since the disease is effectively transmitted to fully immunocompetent recipient mice only if they express CD1d, a non-classical MHC protein that presents lipid antigens to iNKT cells. Molecular analysis revealed that TET2 and TET3 drive iNKT lineage specific DNA demethylation and control gene expression via DNA demethylation and/or chromatin accessibility of key lineage specifying factors such as RORgt, Tbet and ThPOK. Collectively, TET2 and TET3 are fundamental regulators that seal iNKT lineage fate, ensure proper development and maturation and safeguard aberrant TCR mediated expansion.