Aire-expressing medullary thymic epithelial cells (mTECs) play a key role in preventing autoimmunity by expressing tissue-restricted antigens to help purge the emerging T cell receptor repertoire of self-reactive specificities. Here we demonstrate a novel role for a CD4+3− inducer cell population, previously linked to development of organized secondary lymphoid structures and maintenance of T cell memory in the functional regulation of Aire-mediated promiscuous gene expression in the thymus. CD4+3− cells are closely associated with mTECs in adult thymus, and in fetal thymus their appearance is temporally linked with the appearance of Aire+ mTECs. We show that RANKL signals from this cell promote the maturation of RANK-expressing CD80−Aire− mTEC progenitors into CD80+Aire+ mTECs, and that transplantation of RANK-deficient thymic stroma into immunodeficient hosts induces autoimmunity. Collectively, our data reveal cellular and molecular mechanisms leading to the generation of Aire+ mTECs and highlight a previously unrecognized role for CD4+3−RANKL+ inducer cells in intrathymic self-tolerance.

A key role of the thymic medulla is to negatively select autoreactive CD4+ and CD8+ thymocytes, a process important for T cell tolerance induction. However, the involvement of the thymic medulla in other aspects of αβ T cell development, including the generation of Foxp3+ natural regulatory T cells (nTreg cells) and the continued maturation of positively selected conventional αβ T cells, is unclear. We show that newly generated conventional CD69+Qa2− CD4 single-positive thymocytes mature to the late CD69−Qa2+ stage in the absence of RelB-dependent medullary thymic epithelial cells (mTECs). Furthermore, an increasing ability to continue maturation extrathymically is observed within the CD69+CCR7−/loCCR9+ subset of conventional SP4 thymocytes, providing evidence for an independence from medullary support by the earliest stages after positive selection. In contrast, Foxp3+ nTreg cell development is medullary dependent, with mTECs fostering the generation of Foxp3−CD25+ nTreg cell precursors at the CD69+CCR7+CCR9− stage. Our results demonstrate a differential requirement for the thymic medulla in relation to CD4 conventional and Foxp3+ thymocyte lineages, in which an intact mTEC compartment is a prerequisite for Foxp3+ nTreg cell development through the generation of Foxp3−CD25+ nTreg cell precursors.

In the thymus, interactions between immature thymocytes and thymic epithelial cells (TECs) regulate the development and selection of self-tolerant MHC-restricted T cells. Despite the importance of cortical (cTEC) and medullary (mTEC) thymic epithelial cells in fostering T cell production, events in TEC development are still unclear. Although precursor-product relationships during mTEC development have been reported, and some genetic regulators of mTEC development have been identified, stages in cTEC development occurring downstream of recently identified bipotent cTEC/mTEC progenitors remain poorly defined. In this study, we combine analysis of differentiation, proliferation, and gene expression of TECs in the murine thymus, that has enabled us to identify cTEC progenitors, define multiple stages in cTEC development, and identify novel checkpoints in development of the cTEC lineage. We show an essential requirement for FoxN1 in the initial development of cTEC from bipotent progenitors, and demonstrate a stage-specific requirement for CD4−8− thymocytes in later stages of cTEC development. Collectively, our data establish a program of cTEC development that should provide insight into the formation and function of the thymic cortex for T cell development.

Material

SummaryThe thymic medulla provides a specialized microenvironment for the negative selection of T cells, with the presence of autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) during the embryonic-neonatal period being both necessary and sufficient to establish long-lasting tolerance. Here we showed that emergence of the first cohorts of Aire+ mTECs at this key developmental stage, prior to αβ T cell repertoire selection, was jointly directed by Rankl+ lymphoid tissue inducer cells and invariant Vγ5+ dendritic epidermal T cell (DETC) progenitors that are the first thymocytes to express the products of gene rearrangement. In turn, generation of Aire+ mTECs then fostered Skint-1-dependent, but Aire-independent, DETC progenitor maturation and the emergence of an invariant DETC repertoire. Hence, our data attributed a functional importance to the temporal development of Vγ5+ γδ T cells during thymus medulla formation for αβ T cell tolerance induction and demonstrated a Rank-mediated reciprocal link between DETC and Aire+ mTEC maturation.

In the thymus, interactions with both cortical and medullary microenvironments regulate the development of self-tolerant conventional CD4+ and CD8+ αβT cells expressing a wide range of αβTCR specificities. Additionally, the cortex is also required for the development of invariant NKT (iNKT) cells, a specialized subset of T cells that expresses a restricted αβTCR repertoire and is linked to the regulation of innate and adaptive immune responses. Although the role of the cortex in this process is to enable recognition of CD1d molecules expressed by CD4+CD8+ thymocyte precursors, the requirements for additional thymus microenvironments during iNKT cell development are unknown. In this study, we reveal a role for medullary thymic epithelial cells (mTECs) during iNKT cell development in the mouse thymus. This requirement for mTECs correlates with their expression of genes required for IL-15 trans-presentation, and we show that soluble IL-15/IL-15Rα complexes restore iNKT cell development in the absence of mTECs. Furthermore, mTEC development is abnormal in iNKT cell–deficient mice, and early stages in iNKT cell development trigger receptor activator for NF-κB ligand–mediated mTEC development. Collectively, our findings demonstrate that intrathymic iNKT cell development requires stepwise interactions with both the cortex and the medulla, emphasizing the importance of thymus compartmentalization in the generation of both diverse and invariant αβT cells. Moreover, the identification of a novel requirement for iNKT cells in thymus medulla development further highlights the role of both innate and adaptive immune cells in thymus medulla formation.

The thymus supports the production of self-tolerant T cells from immature precursors. Studying the mechanisms regulating the establishment and maintenance of stromal microenvironments within the thymus therefore is essential to our understanding of T-cell production and ultimately immune system functioning. Despite our ability to phenotypically define stromal cell compartments of the thymus, the mechanisms regulating their development and the ways by which they influence T-cell precursors are still unclear. Here, we review recent findings and highlight unresolved issues relating to the development and functioning of thymic stromal cells.

The thymus provides a specialized site for the production of T cells capable of recognizing foreign antigens in the context of self-major histocompatibility complex (MHC) molecules. During development, the thymus arises from an epithelial rudiment containing bipotent progenitors that differentiate into distinct cortical and medullary epithelial cells to regulate the maturation and selection of self-tolerant CD4+ and CD8+ T cells. In addition to their differentiation, thymic epithelial cells undergo cellular expansion to ensure that sufficient intrathymic cellular niches are available to support the large number of immature thymocytes required to form a self-tolerant T-cell pool. Thus, intrathymic T-cell production is intimately linked to the formation and availability of niches within thymic microenvironments. Here, we show the increase in intrathymic niches caused by the proliferation of the epithelium in the developing thymus is temporally regulated, and correlates with the presence of a population of fetal thymic mesenchyme defined by platelet-derived growth factor receptor alpha (PDGFRalpha) expression. Depletion of PDGFRalpha+ mesenchyme from embryonic thymi prior to their transplantation to ectopic sites results in the formation of functional yet hypoplastic thymic tissue. In summary, we highlight a specialized role for PDGFRalpha+ fetal mesenchyme in the thymus by determining availability of thymic niches through the regulation of thymic epithelial proliferation.