Developmentally regulated availability of RANKL and CD40 ligand reveals distinct mechanisms of fetal and adult cross-talk in the thymus medulla.

T cell tolerance in the thymus is a key step in shaping the developing T cell repertoire. Thymic medullary epithelial cells play multiple roles in this process, including negative selection of autoreactive thymocytes, influencing thymic dendritic cell positioning, and the generation of Foxp3(+) regulatory T cells. Previous studies show that medullary thymic epithelial cell (mTEC) development involves hemopoietic cross-talk, and numerous TNFR superfamily members have been implicated in this process. Whereas CD40 and RANK represent key examples, interplay between these receptors, and the individual cell types providing their ligands at both fetal and adult stages of thymus development, remain unclear. In this study, by analysis of the cellular sources of receptor activator for NF-κB ligand (RANKL) and CD40L during fetal and adult cross-talk in the mouse, we show that the innate immune cell system drives initial fetal mTEC development via expression of RANKL, but not CD40L. In contrast, cross-talk involving the adaptive immune system involves both RANKL and CD40L, with analysis of distinct subsets of intrathymic CD4(+) T cells revealing a differential contribution of CD40L by conventional, but not Foxp3(+) regulatory, T cells. We also provide evidence for a stepwise involvement of TNFRs in mTEC development, with CD40 upregulation induced by initial RANK signaling subsequently controlling proliferation within the mTEC compartment. Collectively, our findings show how multiple hemopoietic cell types regulate mTEC development through differential provision of RANKL/CD40L during ontogeny, revealing molecular differences in fetal and adult hemopoietic cross-talk. They also suggest a stepwise process of mTEC development, in which RANK is a master player in controlling the availability of other TNFR family members.