| Abstract: | Induction of self-tolerance in developing T cells depends on medullary thymic epithelial cells (mTECs), whose development, in turn, requires signals from single-positive (SP) thymocytes. Thus, the absence of SP thymocytes in Tcra−/− mice results in a profound deficiency in mTECs. Here, we have probed the mechanism that underlies this requirement for cross-talk with thymocytes in medullary development. Previous studies have implicated nonclassical NF-κB as a pathway important in the development of mTECs, because mice lacking RelB, NIK, or IKKα, critical components of this pathway, have an almost complete absence of mTECs, with resulting autoimmune pathology. We therefore assessed the effect of selective deletion in TEC of TNF receptor-associated factor 3 (TRAF3), an inhibitor of nonclassical NF-κB signaling. Deletion of TRAF3 in thymic epithelial cells allowed RelB-dependent development of normal numbers of AIRE-expressing mTECs in the complete absence of SP thymocytes. Thus, mTEC development can occur in the absence of cross-talk with SP thymocytes, and signals provided by SP T cells are needed to overcome TRAF3-imposed arrest in mTEC development mediated by inhibition of nonclassical NF-κB. We further observed that TRAF3 deletion is also capable of overcoming all requirements for LTβR and CD40, which are otherwise necessary for mTEC development, but is not sufficient to overcome the requirement for RANKL, indicating a role for RANKL that is distinct from the signals provided by SP thymocytes. We conclude that TRAF3 plays a central role in regulation of mTEC development by imposing requirements for SP T cells and costimulation-mediated cross-talk in generation of the medullary compartment.A major role of the thymus is the generation of a functional T-cell repertoire that is broadly responsive to foreign antigens but is self-tolerant. Through their role in exposing developing thymocytes to a spectrum of self-antigens, the stromal cells of the thymus are integral to this tolerization. Of particular importance in this process are the epithelial cells comprising the thymic medulla, the region of the thymus where thymocytes selected into the CD4 and CD8 single-positive (SP) lineages reside before emigrating to the periphery (reviewed in refs. 1–3). The importance of thymic medullary epithelial cells (mTECs) in the maintenance of self-tolerance is illustrated by the destructive autoreactivity that results from disruption of mTEC development (reviewed in ref. 4).Just as mTECs have a central role in shaping the developing T-cell repertoire, thymocytes, in turn, are vital to the development and maintenance of the mTEC compartment, a bidirectional interaction that has been termed cross-talk (5–7). A number of recent reports have characterized the CD4 SP thymocyte–stromal cell interactions that are critical for mTEC development (5–11). However, the mechanism that enforces the requirement for SP thymocytes in mTEC development has not been fully identified.We therefore addressed the signaling requirements that mediate the cross-talk required for mTEC development. Previous studies have implicated nonclassical NF-κB as a pathway important in the development of mTECs. It has been shown that mice lacking RelB, NIK, or IKKα, components of the nonclassical NF-κB pathway, have an almost complete absence of mTECs and exhibit resulting autoimmune pathology (12–16). Engagement of TNF receptor (TNFR) family members including CD40, LTβR, and RANK has been shown to activate nonclassical NF-κB signaling via a pathway regulated by several members of the TRAF family of adaptor/ubiquitin ligase proteins. TRAF3 has a unique role in inhibiting nonclassical NF-κB signaling in resting cells (17), and it has been demonstrated that deletion of TRAF3 in B cells results in constitutive activation of the alternative NF-κB pathway (18, 19). We therefore tested the possibility that TRAF3-mediated inhibition of alternative NF-κB is responsible for the failure of mTEC development in the absence of signals from SP thymocytes. We made the striking observation that deletion of TRAF3 in thymic epithelium is sufficient to allow RelB-dependent mTEC development in the complete absence of TCRαβ SP thymocytes. TRAF3 deletion is also capable of overcoming all requirements for LTβR and CD40 during mTEC development, but is not sufficient to overcome the requirement for RANKL, indicating an essential role for RANKL that is distinct from the signals provided by SP thymocytes. Together, these results demonstrate that mTECs can develop in the complete absence of SP thymocytes and that TRAF3 plays a critical role in imposing the requirement for cross-talk with SP thymocytes, thus linking the appearance of mature thymocytes to the development of the thymic medullary environment necessary for imposing self-tolerance. |
