Lymphotoxin-alpha produced by autoreactive CD4+thymocytes controls formation and homeostasis of the thymic medulla

Introduction The thymus ensures the generation of a diverse repertoire of T lymphocytes capable of mounting immune responses directed against pathogens while avoiding autoimmune attacks directed at self-antigens. The thymic medulla provides a specialized microenvironment dedicated to purging the T-cell repertoire of potentially hazardous self-reactive specificities, and thus plays a pivotal role in preventing the development of autoimmune disorders. Medullary thymic epithelial cells (mTECs) play a pivotal role in establishing T-cell tolerance via their unique capacity to express thousands of peripheral tissue-restricted self-antigens. Conversely, formation of the medulla is controlled by the development of single-positive (SP) thymocytes. However, the precise identities of the thymocyte subset and cytokines involved in this process remain poorly documented. Methods By means of knock-out mice lacking CD4+ or CD8+ SP thymocytes, T cell receptor (TCR)-transgenic mice and in vitro reaggregated thymic organ culture experiments, we have studied the respective contributions of SP CD4+ and CD8+ thymocytes in medulla formation. We have also studied by using fetal thymic organ culture (FTOC) experiments the role of three members of the tumor necrosis factor (TNF) superfamily, namely lymphotoxin (LT), RANK ligand (RANKL) and CD40 ligand (CD40L), in mTEC development and homeostasis. Results We found that SP CD4+ thymocytes are indispensable and sufficient for inducing proper medulla formation. In contrast, CD8+ SP thymocytes are much less efficient and dispensable for sustaining this process. Furthermore, TCR reactivity of SP CD4+ thymocytes with self-antigens expressed by mTECs is compulsory for mTEC development and homeostasis. Importantly, we found that these antigen-dependent interactions with mTECs induce the expression of LTa in autoreactive CD4+thymocytes and the RANKL receptor in mTECs. By using FTOC experiments, we found that of the three individual TNF stimuli (LT, RANKL and CD40L) only the LT induces a significant increase in mTEC cellularity. Remarkably, a synergistic increase in mTEC numbers was observed when LT was combined with RANKL and CD40L. Therefore, LTa expression by autoreactive CD4+thymocytes controls mTEC cellularity by acting in synergy with RANKL and CD40L. Conclusion Our study demonstrates that antigen-dependent interactions between SP CD4+ thymocytes and mTECs finely regulate the expression of LTa in autoreactive CD4+ thymocytes and RANK in mTECs, thereby completing the signaling axes that are critical for mTEC development and homeostasis, processes pivotal for T-cell tolerance induction.