RANK links thymic regulatory T cells to fetal loss and gestational diabetes in pregnancy.

Successful pregnancies rely on adaptations within the mother1, including marked changes within the immune system2. It has long been known that the thymus, the central lymphoid organ, changes markedly during pregnancy3. However, the molecular basis and importance of this process remain largely obscure. Here we show that the osteoclast differentiation receptor RANK4,5 couples female sex hormones to the rewiring of the thymus during pregnancy. Genetic deletion of Rank (also known as Tnfrsf11a) in thymic epithelial cells results in impaired thymic involution and blunted expansion of natural regulatory T (Treg) cells in pregnant female mice. Sex hormones, in particular progesterone, drive the development of thymic Treg cells through RANK in a manner that depends on AIRE+ medullary thymic epithelial cells. The depletion of Rank in the mouse thymic epithelium results in reduced accumulation of natural Treg cells in the placenta, and an increase in the number of miscarriages. Thymic deletion of Rank also results in impaired accumulation of Treg cells in visceral adipose tissue, and is associated with enlarged adipocyte size, tissue inflammation, enhanced maternal glucose intolerance, fetal macrosomia, and a long-lasting transgenerational alteration in glucose homeostasis, which are all key hallmarks of gestational diabetes. Transplantation of Treg cells rescued fetal loss, maternal glucose intolerance and fetal macrosomia. In human pregnancies, we found that gestational diabetes also correlates with a reduced number of Treg cells in the placenta. Our findings show that RANK promotes the hormone-mediated development of thymic Treg cells during pregnancy, and expand the functional role of maternal Treg cells to the development of gestational diabetes and the transgenerational metabolic rewiring of glucose homeostasis.

Assembling the thymus medulla: Development and function of epithelial cell heterogeneity.

The thymus is a unique primary lymphoid organ that supports the production of self-tolerant T-cells essential for adaptive immunity. Intrathymic microenvironments are microanatomically compartmentalised, forming defined cortical, and medullary regions each differentially supporting critical aspects of thymus-dependent T-cell maturation. Importantly, the specific functional properties of thymic cortical and medullary compartments are defined by highly specialised thymic epithelial cells (TEC). For example, in the medulla heterogenous medullary TEC (mTEC) contribute to the enforcement of central tolerance by supporting deletion of autoreactive T-cell clones, thereby counterbalancing the potential for random T-cell receptor generation to contribute to autoimmune disease. Recent advances have further shed light on the pathways and mechanisms that control heterogeneous mTEC development and how differential mTEC functionality contributes to control self-tolerant T-cell development. Here we discuss recent findings in relation to mTEC development and highlight examples of how mTEC diversity contribute to thymus medulla function.

The medulla controls effector primed γδT-cell development in the adult mouse thymus.

γδT cells are produced in the thymus throughout life and provide immunity at epithelial-rich sites. Unlike conventional αßT cells, γδT-cell development involves intrathymic acquisition of effector function, with priming for either IL17 or IFN-γ production occurring during embryonic or adult life, respectively. How the thymus controls effector-primed γδT-cell generation in adulthood is poorly understood. Here, we distinguished de novo γδT cells from those undergoing thymus recirculation and/or retention using Rag2GFP mice alongside markers of maturation/effector priming including CD24, CD25, CD73, and IFN-γ, the latter by crossing with IFN-γYFP GREAT mice. We categorize newly developing γδT-cells into an ordered sequence where CD25+ CD73- IFN-γYFP- precursors are followed sequentially by CD25- CD73+ IFN-γYFP- intermediates and CD25- CD73+ IFN-γYFP+ effectors. To determine intrathymic requirements controlling this sequence, we examined γδT-cell development in Relb-/- thymus grafts that lack medullary microenvironments. Interestingly, medulla deficiency did not alter CD25+ γδT-cell precursor generation, but significantly impaired development of effector primed stages. This impact on γδT-cell priming was mirrored in plt/plt mice lacking the medullary chemoattractants CCL19 and CCL21, and also Ccl21a-/- but not Ccl19-/- mice. Collectively, we identify the medulla as an important site for effector priming during adult γδT-cell development and demonstrate a specific role for the medullary epithelial product CCL21 in this process.

Diversity in Cortical Thymic Epithelial Cells Occurs through Loss of a Foxn1-Dependent Gene Signature Driven by Stage-Specific Thymocyte Cross-Talk.

In the thymus, cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells support αßT cell development from lymphoid progenitors. For cTECs, expression of a specialized gene signature that includes Cxcl12, Dll4, and Psmb11 enables the cortex to support T lineage commitment and the generation and selection of CD4+CD8+ thymocytes. Although the importance of cTECs in T cell development is well defined, mechanisms that shape the cTEC compartment and regulate its functional specialization are unclear. Using a Cxcl12DsRed reporter mouse model, we show that changes in Cxcl12 expression reveal a developmentally regulated program of cTEC heterogeneity. Although cTECs are uniformly Cxcl12DsRed+ during neonatal stages, progression through postnatal life triggers the appearance of Cxcl12DsRed- cTECs that continue to reside in the cortex alongside their Cxcl12DsRed+ counterparts. This appearance of Cxcl12DsRed- cTECs is controlled by maturation of CD4-CD8-, but not CD4+CD8+, thymocytes, demonstrating that stage-specific thymocyte cross-talk controls cTEC heterogeneity. Importantly, although fate-mapping experiments show both Cxcl12DsRed+ and Cxcl12DsRed- cTECs share a common Foxn1+ cell origin, RNA sequencing analysis shows Cxcl12DsRed- cTECs no longer express Foxn1, which results in loss of the FOXN1-dependent cTEC gene signature and may explain the reduced capacity of Cxcl12DsRed- cTECs for thymocyte interactions. In summary, our study shows that shaping of the cTEC compartment during the life course occurs via stage-specific thymocyte cross-talk, which drives loss of Foxn1 expression and its key target genes, which may then determine the functional competence of the thymic cortex.

Diversity in Cortical Thymic Epithelial Cells Occurs through Loss of a Foxn1-Dependent Gene Signature Driven by Stage-Specific Thymocyte Cross-Talk.

In the thymus, cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells support αßT cell development from lymphoid progenitors. For cTECs, expression of a specialized gene signature that includes Cxcl12, Dll4, and Psmb11 enables the cortex to support T lineage commitment and the generation and selection of CD4+CD8+ thymocytes. Although the importance of cTECs in T cell development is well defined, mechanisms that shape the cTEC compartment and regulate its functional specialization are unclear. Using a Cxcl12 DsRed reporter mouse model, we show that changes in Cxcl12 expression reveal a developmentally regulated program of cTEC heterogeneity. Although cTECs are uniformly Cxcl12 DsRed+ during neonatal stages, progression through postnatal life triggers the appearance of Cxcl12 DsRed- cTECs that continue to reside in the cortex alongside their Cxcl12 DsRed+ counterparts. This appearance of Cxcl12 DsRed- cTECs is controlled by maturation of CD4-CD8-, but not CD4+CD8+, thymocytes, demonstrating that stage-specific thymocyte cross-talk controls cTEC heterogeneity. Importantly, although fate-mapping experiments show both Cxcl12 DsRed+ and Cxcl12 DsRed- cTECs share a common Foxn1 + cell origin, RNA sequencing analysis shows Cxcl12 DsRed- cTECs no longer express Foxn1, which results in loss of the FOXN1-dependent cTEC gene signature and may explain the reduced capacity of Cxcl12 DsRed- cTECs for thymocyte interactions. In summary, our study shows that shaping of the cTEC compartment during the life course occurs via stage-specific thymocyte cross-talk, which drives loss of Foxn1 expression and its key target genes, which may then determine the functional competence of the thymic cortex.

A novel method to identify Post-Aire stages of medullary thymic epithelial cell differentiation.

Autoimmune regulator+ (Aire) medullary thymic epithelial cells (mTECs) play a critical role in tolerance induction. Several studies demonstrated that Aire+ mTECs differentiate further into Post-Aire cells. Yet, the identification of terminal stages of mTEC maturation depends on unique fate-mapping mouse models. Herein, we resolve this limitation by segmenting the mTEChi (MHCIIhi CD80hi ) compartment into mTECA/hi (CD24- Sca1- ), mTECB/hi (CD24+ Sca1- ), and mTECC/hi (CD24+ Sca1+ ). While mTECA/hi included mostly Aire-expressing cells, mTECB/hi contained Aire+ and Aire- cells and mTECC/hi were mainly composed of cells lacking Aire. The differential expression pattern of Aire led us to investigate the precursor-product relationship between these subsets. Strikingly, transcriptomic analysis of mTECA/hi , mTECB/hi , and mTECC/hi sequentially mirrored the specific genetic program of Early-, Late- and Post-Aire mTECs. Corroborating their Post-Aire nature, mTECC/hi downregulated the expression of tissue-restricted antigens, acquired traits of differentiated keratinocytes, and were absent in Aire-deficient mice. Collectively, our findings reveal a new and simple blueprint to survey late stages of mTEC differentiation.

Rank signaling links the development of invariant γδ T cell progenitors and Aire(+) medullary epithelium.

The 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 αß 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 αß T cell tolerance induction and demonstrated a Rank-mediated reciprocal link between DETC and Aire(+) mTEC maturation.

Invariant NKT Cells and Control of the Thymus Medulla.

Most αß T cells that form in the thymus are generated during mainstream conventional thymocyte development and involve the generation and selection of a diverse αß TCR repertoire that recognizes self-peptide/MHC complexes. Additionally, the thymus also supports the production of T cell subsets that express αß TCRs but display unique developmental and functional features distinct from conventional αß T cells. These include multiple lineages of CD1d-restricted invariant NKT (iNKT) cells that express an invariant αß TCR, branch off from mainstream thymocytes at the CD4+CD8+ stage, and are potent producers of polarizing cytokines. Importantly, and despite their differences, iNKT cells and conventional αß T cells share common requirements for thymic epithelial microenvironments during their development. Moreover, emerging evidence suggests that constitutive cytokine production by iNKT cells influences both conventional thymocyte development and the intrathymic formation of additional innate CD8+ αß T cells with memory-like properties. In this article, we review evidence for an intrathymic innate lymphocyte network in which iNKT cells play key roles in multiple aspects of thymus function.

Retinoic Acid Signaling in Thymic Epithelial Cells Regulates Thymopoiesis.

Despite the essential role of thymic epithelial cells (TEC) in T cell development, the signals regulating TEC differentiation and homeostasis remain incompletely understood. In this study, we show a key in vivo role for the vitamin A metabolite, retinoic acid (RA), in TEC homeostasis. In the absence of RA signaling in TEC, cortical TEC (cTEC) and CD80loMHC class IIlo medullary TEC displayed subset-specific alterations in gene expression, which in cTEC included genes involved in epithelial proliferation, development, and differentiation. Mice whose TEC were unable to respond to RA showed increased cTEC proliferation, an accumulation of stem cell Ag-1hi cTEC, and, in early life, a decrease in medullary TEC numbers. These alterations resulted in reduced thymic cellularity in early life, a reduction in CD4 single-positive and CD8 single-positive numbers in both young and adult mice, and enhanced peripheral CD8+ T cell survival upon TCR stimulation. Collectively, our results identify RA as a regulator of TEC homeostasis that is essential for TEC function and normal thymopoiesis.

Control of the thymic medulla and its influence on αßT-cell development.

The thymus is a primary lymphoid tissue that supports the generation of αßT cells. In this review, we describe the processes that give rise to the thymus medulla, a site that nurtures self-tolerant T-cell generation following positive selection events that take place in the cortex. To summarize the developmental pathways that generate medullary thymic epithelial cells (mTEC) from their immature progenitors, we describe work on both the initial emergence of the medulla during embryogenesis, and the maintenance of the medulla during postnatal stages. We also investigate the varying roles that receptors belonging to the tumor necrosis factor receptor superfamily have on thymus medulla development and formation, and highlight the impact that T-cell development has on thymus medulla formation. Finally, we examine the evidence that the thymic medulla plays an important role during the intrathymic generation of distinct αßT-cell subtypes. Collectively, these studies provide new insight into the development and functional importance of medullary microenvironments during self-tolerant T-cell production in the thymus.

Dynamic changes in intrathymic ILC populations during murine neonatal development.

Members of the innate lymphoid cell (ILC) family have been implicated in the development of thymic microenvironments and the recovery of this architecture after damage. However, a detailed characterization of this family in the thymus is lacking. To better understand the thymic ILC compartment, we have utilized multiple in vivo models including the fate mapping of inhibitor of DNA binding-2 (Id2) expression and the use of Id2 reporter mice. Our data demonstrate that ILCs are more prominent immediately after birth, but were rapidly diluted as the T-cell development program increased. As observed in the embryonic thymus, CCR6+ NKp46- lymphoid tissue inducer (LTi) cells were the main ILC3 population present, but numbers of these cells swiftly declined in the neonate and ILC3 were barely detectable in adult thymus. This loss of ILC3 means ILC2 are the dominant ILC population in the thymus. Thymic ILC2 were able to produce IL-5 and IL-13, were located within the medulla, and did not result from ILC3 plasticity. Furthermore, in WT mice, thymic ILC2 express little RANKL (receptor activator of nuclear factor kappa-B ligand) arguing that functionally, these cells provide different signals to LTi cells in the thymus. Collectively, these data reveal a dynamic switch in the ILC populations of the thymus during neonatal development.

Aire controls the recirculation of murine Foxp3+ regulatory T-cells back to the thymus.

In the thymus, medullary thymic epithelial cells (mTEC) determine the fate of newly selected CD4+ and CD8+ single positive (SP) thymocytes. For example, mTEC expression of Aire controls intrathymic self-antigen availability for negative selection. Interestingly, alterations in both Foxp3+ Regulatory T-cells (T-Reg) and conventional SP thymocytes in Aire-/- mice suggest additional, yet poorly understood, roles for Aire during intrathymic T-cell development. To examine this, we analysed thymocytes from Aire-/- mice using Rag2GFP and Foxp3 expression, and a recently described CD69/MHCI subset definition of post-selection CD4+ conventional thymocytes. We show that while Aire is dispensable for de novo generation of conventional αßT-cells, it plays a key role in controlling the intrathymic T-Reg pool. Surprisingly, a decline in intrathymic T-Reg in Aire-/- mice maps to a reduction in mature recirculating Rag2GFP- T-Reg that express CCR6 and re-enter the thymus from the periphery. Furthermore, we show mTEC expression of the CCR6 ligand CCL20 is reduced in Aire-/- mice, and that CCR6 is required for T-Reg recirculation back to the thymus. Collectively, our study re-defines requirements for late stage intrathymic αßT-cell development, and demonstrates that Aire controls a CCR6-CCL20 axis that determines the developmental makeup of the intrathymic T-Reg pool.

Osteoprotegerin-Mediated Homeostasis of Rank+ Thymic Epithelial Cells Does Not Limit Foxp3+ Regulatory T Cell Development.

In the thymus, medullary thymic epithelial cells (mTEC) regulate T cell tolerance via negative selection and Foxp3(+) regulatory T cell (Treg) development, and alterations in the mTEC compartment can lead to tolerance breakdown and autoimmunity. Both the receptor activator for NF-κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) axis and expression of the transcriptional regulator Aire are involved in the regulation of thymus medullary microenvironments. However, their impact on the mechanisms controlling mTEC homeostasis is poorly understood, as are the processes that enable the thymus medulla to support the balanced production of mTEC-dependent Foxp3(+) Treg. In this study, we have investigated the control of mTEC homeostasis and examined how this process impacts the efficacy of Foxp3(+) Treg development. Using newly generated RANK Venus reporter mice, we identify distinct RANK(+) subsets that reside within both the mTEC(hi) and mTEC(lo) compartments and that represent direct targets of OPG-mediated control. Moreover, by mapping OPG expression to a subset of Aire(+) mTEC, our data show how cis- and trans-acting mechanisms are able to control the thymus medulla by operating on multiple mTEC targets. Finally, we show that whereas the increase in mTEC availability in OPG-deficient (Tnfrsf11b(-/-)) mice impacts the intrathymic Foxp3(+) Treg pool by enhancing peripheral Treg recirculation back to the thymus, it does not alter the number of de novo Rag2pGFP(+)Foxp3(+) Treg that are generated. Collectively, our study defines patterns of RANK expression within the thymus medulla, and it shows that mTEC homeostasis is not a rate-limiting step in intrathymic Foxp3(+) Treg production.

CCRL1/ACKR4 is expressed in key thymic microenvironments but is dispensable for T lymphopoiesis at steady state in adult mice.

Thymus colonisation and thymocyte positioning are regulated by interactions between CCR7 and CCR9, and their respective ligands, CCL19/CCL21 and CCL25. The ligands of CCR7 and CCR9 also interact with the atypical receptor CCRL1 (also known as ACKR4), which is expressed in the thymus and has recently been reported to play an important role in normal αßT-cell development. Here, we show that CCRL1 is expressed within the thymic cortex, predominantly by MHC-II(low) CD40(-) cortical thymic epithelial cells and at the subcapsular zone by a population of podoplanin(+) thymic epithelial cells in mice. Interestingly, CCRL1 is also expressed by stromal cells which surround the pericytes of vessels at the corticomedullary junction, the site for progenitor cell entry and mature thymocyte egress from the thymus. We show that CCRL1 suppresses thymocyte progenitor entry into the thymus, however, the thymus size and cellularity are the same in adult WT and CCRL1(-/-) mice. Moreover, CCRL1(-/-) mice have no major perturbations in T-cell populations at different stages of thymic differentiation and development, and have a similar rate of thymocyte migration into the blood. Collectively, our findings argue against a major role for CCRL1 in normal thymus development and function.

An essential role for medullary thymic epithelial cells during the intrathymic development of invariant NKT cells.

In the thymus, interactions with both cortical and medullary microenvironments regulate the development of self-tolerant conventional CD4(+) and CD8(+) αßT cells expressing a wide range of αßTCR 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 αßTCR 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 αßT 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.

Differential requirement for CCR4 and CCR7 during the development of innate and adaptive αßT cells in the adult thymus.

αßT cell development depends upon serial migration of thymocyte precursors through cortical and medullary microenvironments, enabling specialized stromal cells to provide important signals at specific stages of their development. Although conventional αßT cells are subject to clonal deletion in the medulla, entry into the thymus medulla also fosters αßT cell differentiation. For example, during postnatal periods, the medulla is involved in the intrathymic generation of multiple αßT cell lineages, notably the induction of Foxp3(+) regulatory T cell development and the completion of invariant NKT cell development. Although migration of conventional αßT cells to the medulla is mediated by the chemokine receptor CCR7, how other T cell subsets gain access to medullary areas during their normal development is not clear. In this study, we show that combining a panel of thymocyte maturation markers with cell surface analysis of CCR7 and CCR4 identifies distinct stages in the development of multiple αßT cell lineages in the thymus. Although Aire regulates expression of the CCR4 ligands CCL17 and CCL22, we show that CCR4 is dispensable for thymocyte migration and development in the adult thymus, demonstrating defective T cell development in Aire(-/-) mice is not because of a loss of CCR4-mediated migration. Moreover, we reveal that CCR7 controls the development of invariant NKT cells by enabling their access to IL-15 trans-presentation in the thymic medulla and influences the balance of early and late intrathymic stages of Foxp3(+) regulatory T cell development. Collectively, our data identify novel roles for CCR7 during intrathymic T cell development, highlighting its importance in enabling multiple αßT cell lineages to access the thymic medulla.

Natural Th17 cells are critically regulated by functional medullary thymic microenvironments.

The thymic medulla is critical for the enforcement of central tolerance. In addition to deletion of auto-reactive T-cells, the thymic medulla supports the maturation of heterogeneous natural αßT-cells linked to tolerance mechanisms. Natural IL-17-secreting CD4(+)αßT-cells (nTh17) represent recently described natural αßT-cells that mature and undergo functional priming intrathymically. Despite a proposed potential to impact upon either protective or pathological inflammatory responses, the intrathymic mechanisms regulating the balance of nTh17 development are unclear. Here we compare the development of distinct natural αßT-cells in the thymus. We reveal that thymic stromal MHC class II expression and RelB-dependent medullary thymic epithelial cells (mTEC), including Aire(+) mTEC, are an essential requirement for nTh17 development. nTh17 demonstrate a partial, non-redundant requirement for both ICOS-ligand and CD80/86 costimulation, with a dispensable role for CD80/86 expression by thymic epithelial cells. Although mTEC constitutively expressed inducible nitric oxide synthase (iNOS), a critical negative regulator of conventional Th17 differentiation, iNOS was not essential to constrain thymic nTh17. These findings highlight the critical role of the thymic medulla in the differential regulation of novel natural αßT-cell subsets, and reveal additional layers of thymic medullary regulation of T-cell driven autoimmunity and inflammation.

Mechanisms of thymus medulla development and function.

The development of CD4(+) helper and CD8(+) cytotoxic T-cells expressing the αß form of the T-cell receptor (αßTCR) takes place in the thymus, a primary lymphoid organ containing distinct cortical and medullary microenvironments. While the cortex represents a site of early T-cell precursor development, and the positive selection of CD4(+)8(+) thymocytes, the thymic medulla plays a key role in tolerance induction, ensuring that thymic emigrants are purged of autoreactive αßTCR specificities. In recent years, advances have been made in understanding the development and function of thymic medullary epithelial cells, most notably the subset defined by expression of the Autoimmune Regulator (Aire) gene. Here, we summarize current knowledge of the developmental mechanisms regulating thymus medulla development, and examine the role of the thymus medulla in recessive (negative selection) and dominant (T-regulatory cell) tolerance.

A novel method to allow noninvasive, longitudinal imaging of the murine immune system in vivo.

In vivo imaging has revolutionized understanding of the spatiotemporal complexity that subserves the generation of successful effector and regulatory immune responses. Until now, invasive surgery has been required for microscopic access to lymph nodes (LNs), making repeated imaging of the same animal impractical and potentially affecting lymphocyte behavior. To allow longitudinal in vivo imaging, we conceived the novel approach of transplanting LNs into the mouse ear pinna. Transplanted LNs maintain the structural and cellular organization of conventional secondary lymphoid organs. They participate in lymphocyte recirculation and exhibit the capacity to receive and respond to local antigenic challenge. The same LN could be repeatedly imaged through time without the requirement for surgical exposure, and the dynamic behavior of the cells within the transplanted LN could be characterized. Crucially, the use of blood vessels as fiducial markers also allowed precise re-registration of the same regions for longitudinal imaging. Thus, we provide the first demonstration of a method for repeated, noninvasive, in vivo imaging of lymphocyte behavior.

Mesenchymal cells regulate retinoic acid receptor-dependent cortical thymic epithelial cell homeostasis.

The vitamin A metabolite and transcriptional modulator retinoic acid (RA) is recognized as an important regulator of epithelial cell homeostasis in several tissues. Despite the known importance of the epithelial compartment of the thymus in T cell development and selection, the potential role of RA in the regulation of thymic cortical and medullary epithelial cell homeostasis has yet to be addressed. In this study, using fetal thymus organ cultures, we demonstrate that endogenous RA signaling promotes thymic epithelial cell (TEC) cell-cycle exit and restricts TEC cellularity preferentially in the cortical TEC compartment. Combined gene expression, biochemical, and functional analyses identified mesenchymal cells as the major source of RA in the embryonic thymus. In reaggregate culture experiments, thymic mesenchyme was required for RA-dependent regulation of TEC expansion, highlighting the importance of mesenchyme-derived RA in modulating TEC turnover. The RA-generating potential of mesenchymal cells was selectively maintained within a discrete Ly51(int)gp38(+) subset of Ly51(+) mesenchyme in the adult thymus, suggesting a continual role for mesenchymal cell-derived RA in postnatal TEC homeostasis. These findings identify RA signaling as a novel mechanism by which thymic mesenchyme influences TEC development.