Sequential phases in the development of Aire-expressing medullary thymic epithelial cells involve distinct cellular input

Intrathymic deletion of immature thymocytes that express self-reactive TCR specificities is essential in the generation of self tolerance. Medullary thymic epithelial cells (mTEC) expressing the transcriptional regulator Aire play a key role in this process by regulating expression of tissue-restricted antigens to ensure tolerance to peripheral tissues. Here, we have analysed the cellular and molecular requirements for the initial appearance of Aire⁺ mTEC in the embryonic thymus, in addition to their persistence in the adult thymus. Analysis of thymic ontogeny shows that the emergence of embryonic Aire⁺ mTEC occurs prior to the appearance of mature thymocytes, and depends upon lymphoid tissue inducer cells expressing retinoic acid receptor-related orphan receptor γ. In the adult thymus, we show that Aire⁺ mTEC develop in the absence of thymocyte positive and negative selection and CD40 signalling, but are present at reduced frequency. Collectively these data support a model where the initial differentiation of Aire⁺ mTEC involves receptor activator of NF-κB (RANK)-RANKL interactions with lymphoid tissue inducer cells, with subsequent mTEC turnover and/or survival involving CD40-mediated signalling following interactions with mature CD4⁺ thymocytes that express CD40L.