Immunological tolerance is essential in preventing autoimmunity. The establishment of T-cell tolerance is induced in the thymus mainly by the deletion of hazardous autoreactive T cells. Medullary thymic epithelial cells (mTECs) are critical in this process because they express a diverse repertoire of peripheral tissue-restricted self-antigens (TRAs). Hazardous developing T-cells recognizing these TRAs with a high affinity are deleted. TRA expression is regulated by a transcription factor: the autoimmune regulator, Aire. I have demonstrated that antigen (Ag)-specific interactions with autoreactive CD4+ thymocytes play a privileged role in governing the differentiation of Aire+ mTECs. Although these cell interactions are of pivotal importance for T-cell tolerance induction, the underlying molecular mechanisms remain to be characterized. This proposal will define the affinity threshold required for driving CD4+ thymocytes mediated Aire+ mTEC differentiation. We will particularly determine whether mTEC differentiation is coupled to deletion of autoreactive CD4+ thymocytes or whether it corresponds to a dissociable event. We will investigate the epigenetic regulation and the gene expression program that drive the differentiation of Aire+ mTECs induced by CD4+ thymocyte interactions. For this, we will take advantage of genetically modified mice in which Ag presentation to CD4+ T cells is selectively abrogated in mTECs. A particular emphasis will be given to the molecular and epigenetic regulation of the Aire gene locus. This work will entail the use of cutting edge techniques in molecular biology (e.g. ChIP-seq or RNA-seq) and in cellular biology (e.g. fetal thymic organoculture) and will involve the use of unique transgenic mouse models. Overall, the combination of these tools will allow us to dissect the molecular mechanisms that drive proper mTEC differentiation, which is essential for ensuring central T-cell tolerance and hence for protecting from autoimmunity.