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.

Rethinking Thymic Tolerance: Lessons from Mice.

In the thymus, distinct cortex and medulla areas emphasize the division of labor in selection events shaping the αßT cell receptor repertoire. For example, MHC restriction via positive selection is a unique property of epithelial cells in the thymic cortex. Far less clear are the events controlling tolerance induction in the medulla. By acting in concert through multiple roles, including antigen production/presentation and chemokine-mediated control of migration, we propose that medullary epithelium and dendritic cells collectively enable the medulla to balance T cell production with negative selection and Foxp3+ regulatory T cell (Treg) development. We examine here the features of these medullary resident cells and their roles in T cell tolerance, and discuss how imbalance in the thymus can result in loss of T cell tolerance.

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.

Formation of the Intrathymic Dendritic Cell Pool Requires CCL21-Mediated Recruitment of CCR7+ Progenitors to the Thymus.

During αß T cell development in the thymus, migration of newly selected CD4+ and CD8+ thymocytes into medullary areas enables tolerance mechanisms to purge the newly selected αß TCR repertoire of autoreactive specificities. Thymic dendritic cells (DC) play key roles in this process and consist of three distinct subsets that differ in their developmental origins. Thus, plasmacytoid DC and Sirpα+ conventional DC type 2 are extrathymically derived and enter into the thymus via their respective expression of the chemokine receptors CCR9 and CCR2. In contrast, although Sirpα- conventional DC type 1 (cDC1) are known to arise intrathymically from immature progenitors, the precise nature of such thymus-colonizing progenitors and the mechanisms controlling their thymus entry are unclear. In this article, we report a selective reduction in thymic cDC1 in mice lacking the chemokine receptor CCR7. In addition, we show that the thymus contains a CD11c+MHC class II-Sirpα-Flt3+ cDC progenitor population that expresses CCR7, and that migration of these cells to the thymus is impaired in Ccr7-/- mice. Moreover, thymic cDC1 defects in Ccr7-/- mice are mirrored in plt/plt mice, with further analysis of mice individually lacking the CCR7 ligands CCL21Ser (Ccl21a-/- ) or CCL19 (Ccl19-/-) demonstrating an essential role for CCR7-CCL21Ser during intrathymic cDC1 development. Collectively, our data support a mechanism in which CCR7-CCL21Ser interactions guide the migration of cDC progenitors to the thymus for correct formation of the intrathymic cDC1 pool.

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.

Lymphotoxin ß Receptor Controls T Cell Progenitor Entry to the Thymus.

The recruitment of lymphoid progenitors to the thymus is essential to sustain T cell production throughout life. Importantly, it also limits T lineage regeneration following bone marrow transplantation, and so contributes to the secondary immunodeficiency that is caused by delayed immune reconstitution. Despite this significance, the mechanisms that control thymus colonization are poorly understood. In this study, we show that in both the steady-state and after bone marrow transplant, lymphotoxin ß receptor (LTßR) controls entry of T cell progenitors to the thymus. We show that this requirement maps to thymic stroma, further underlining the key importance of this TNFR superfamily member in regulation of thymic microenvironments. Importantly, analysis of the requirement for LTßR in relationship to known regulators of thymus seeding suggests that it acts independently of its regulation of thymus-homing chemokines. Rather, we show that LTßR differentially regulates intrathymic expression of adhesion molecules known to play a role in T cell progenitor entry to the thymus. Finally, Ab-mediated in vivo LTßR stimulation following bone marrow transplant enhances initial thymus recovery and boosts donor-derived T cell numbers, which correlates with increased adhesion molecule expression by thymic stroma. Collectively, we reveal a novel link between LTßR and thymic stromal cells in thymus colonization, and highlight its potential as an immunotherapeutic target to boost T cell reconstitution after transplantation.

Relb acts downstream of medullary thymic epithelial stem cells and is essential for the emergence of RANK(+) medullary epithelial progenitors.

Thymic epithelial cells (TECs) provide essential signals for αßT-cell development, and medullary TECs (mTECs) control T-cell tolerance through both negative selection and Foxp3(+) regulatory T (Treg) cell development. Although heterogeneity within the mTEC compartment is well studied, the molecular regulators of specific stages of mTEC development are still poorly understood. Given the importance of the RANK-RANKL axis in thymus medulla formation, we have used RANK Venus reporter mice to analyze the ontogeny of RANK(+) TECs during development and correlated RANK expression with mTEC stem cells defined by SSEA-1. In addition, we have investigated how requirements for the key regulators Foxn1 and Relb map to specific stages of mTEC development. Here, we show SSEA-1(+) mTEC stem cells emerge prior to RANK expression and are present in both nude and Relb(-/-) mice, providing direct evidence that mTEC lineage specification occurs independently of Foxn1 and Relb. In contrast, we show that Relb is necessary for the effective production of downstream RANK(+) mTEC progenitors. Collectively, our work defines stage-specific requirements for critical TEC regulators during medulla development, including the timing of Relb dependency, and provides new information on mechanisms controlling mTEC specification.

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.

The thymus and T-cell commitment: the right niche for Notch?

The current dogma is that the thymus is colonized by progenitors that retain the capacity to generate both T cells and B cells, and that intrathymic Notch signalling determines lineage choice so that T cells, rather than B cells, develop in the thymus. However, evidence is now accumulating to indicate that, at least during fetal life, this is not the case. Rather, it now seems that the fetal thymus is colonized by progenitors that have already made the T-cell versus B-cell lineage choice. We propose an alternative role for Notch signalling in the thymus, which is not to mediate this choice but instead to reveal it by supporting further T-cell differentiation in the thymic microenvironment.

Border control: Anatomical origins of the thymus medulla.

The thymus is an anatomically compartmentalized primary lymphoid organ that fosters the production of self-tolerant T cells. The thymic cortex provides a specialized microenvironment in which cortical thymic epithelial cells (cTECs) support the positive selection and further differentiation of self-MHC-restricted thymocytes. Following their migration into the medulla, positively selected thymocytes are further screened for self-reactivity, which involves both negative selection and Foxp3(+) regulatory T cell generation via interactions with medullary thymic epithelial cells (mTECs). Given the importance of both cortical and medullary microenvironments for T cell development, studies that address the developmental origins of cTECs and mTECs are important in understanding the processes that shape the developing T cell receptor repertoire, and reduce the frequency of self-reactive T cells that initiate autoimmune disease. In this issue of the European Journal of Immunology, Onder et al. [Eur. J. Immunol. 2015. 45: 2218-2231] identified a subset of podoplanin(+) mTECs in mice that reside at the corticomedullary junction (CMJ), show that their development is important to establish self-tolerance, and require the presence of self-reactive T cells. Collectively, their findings highlight the CMJ as a potential repository for precursors of the mTEC lineage, and provide a better understanding of thymus medulla formation.

Thymus medulla fosters generation of natural Treg cells, invariant γδ T cells, and invariant NKT cells: what we learn from intrathymic migration.

The organization of the thymus into distinct cortical and medullary regions enables it to control the step-wise migration and development of immature T-cell precursors. Such a process provides access to specialized cortical and medullary thymic epithelial cells at defined stages of maturation, ensuring the generation of self-tolerant and MHC-restricted conventional CD4(+) and CD8(+) αß T cells. The migratory cues and stromal cell requirements that regulate the development of conventional αß T cells have been well studied. However, the thymus also fosters the generation of several immunoregulatory T-cell populations that form key components of both innate and adaptive immune responses. These include Foxp3(+) natural regulatory T cells, invariant γδ T cells, and CD1d-restricted invariant natural killer T cells (iNKT cells). While less is known about the intrathymic requirements of these nonconventional T cells, recent studies have highlighted the importance of the thymus medulla in their development. Here, we review recent findings on the mechanisms controlling the intrathymic migration of distinct T-cell subsets, and relate this to knowledge of the microenvironmental requirements of these cells.

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.