The thymic medulla provides a microenvironment where medullary thymic epithelial cells (mTECs) express autoimmune regulator and diverse tissue-restricted genes, contributing to launching self-tolerance. Positive selection is essential for thymic medulla formation via a previously unknown mechanism. Here we show that the cytokine RANK ligand (RANKL) was produced by positively selected thymocytes and regulated the cellularity of mTEC by interacting with RANK and osteoprotegerin. Forced expression of RANKL restored thymic medulla in mice lacking positive selection, whereas RANKL perturbation impaired medulla formation. These results indicate that RANKL produced by positively selected thymocytes is responsible for fostering thymic medulla formation, thereby establishing central tolerance.

Medullary thymic epithelial cells (mTECs) establish T cell self-tolerance through the expression of autoimmune regulator (Aire) and peripheral tissue-specific self-antigens. However, signals underlying mTEC development remain largely unclear. Here, we demonstrate crucial regulation of mTEC development by receptor activator of NF-kappaB (RANK) and CD40 signals. Whereas only RANK signaling was essential for mTEC development during embryogenesis, in postnatal mice, cooperation between CD40 and RANK signals was required for mTEC development to successfully establish the medullary microenvironment. Ligation of RANK or CD40 on fetal thymic stroma in vitro induced mTEC development in a tumor necrosis factor-associated factor 6 (TRAF6)-, NF-kappaB inducing kinase (NIK)-, and IkappaB kinase beta (IKKbeta)-dependent manner. These results show that developmental-stage-dependent cooperation between RANK and CD40 promotes mTEC development, thereby establishing self-tolerance.

The microenvironments of the thymus are generated by thymic epithelial cells (TECs) and are essential for inducing immune self-tolerance or developing T cells. However, the molecular mechanisms that underlie the differentiation of TECs and thymic compartmentalization are not fully understood. Here we show that deficiency in the tumor necrosis factor receptor-associated factor (TRAF) 6 results in disorganized distribution of medullary TECs (mTECs) and the absence of mature mTECs. Engraftment of thymic stroma of TRAF6(-/-) embryos into athymic nude mice induced autoimmunity. Thus, TRAF6 directs the development of thymic stroma and represents a critical point of regulation for self-tolerance and autoimmunity.