Song Baik

Generation of both cortical and Aire+ medullary thymic epithelial compartments from CD205+ progenitors

In the adult thymus, the development of self‐tolerant thymocytes requires interactions with thymic epithelial cells (TECs). Although both cortical and medullary TECs (cTECs/mTECs) are known to arise from common bipotent TEC progenitors, the phenotype of these progenitors and the timing of the emergence of these distinct lineages remain unclear. Here, we have investigated the phenotype and developmental properties of bipotent TEC progenitors during cTEC/mTEC lineage development. We show that TEC progenitors can undergo a stepwise acquisition of first cTEC and then mTEC hallmarks, resulting in the emergence of a progenitor population simultaneously expressing the cTEC marker CD205 and the mTEC regulator Receptor Activator of NF‐κB (RANK). In vivo analysis reveals the capacity of CD205+ TECs to generate functionally competent cortical and medullary microenvironments containing both cTECs and Aire+ mTECs. Thus, TEC development involves a stage in which bipotent progenitors can co‐express hallmarks of the cTEC and mTEC lineages through sequential acquisition, arguing against a simple binary model in which both lineages diverge simultaneously from bipotent lineage negative TEC progenitors. Rather, our data reveal an unexpected overlap in the phenotypic properties of these bipotent TECs with their lineage‐restricted counterparts.

Serial progression of cortical and medullary thymic epithelial microenvironments.

Thymic epithelial cells (TECs) provide key instructive signals for T‐cell differentiation. Thymic cortical (cTECs) and medullary (mTECs) epithelial cells constitute two functionally distinct microenvironments for T‐cell development, which derive from a common bipotent TEC progenitor. While seminal studies have partially elucidated events downstream of bipotent TECs in relation to the emergence of mTECs and their progenitors, the control and timing of the emergence of the cTEC lineage, particularly in relation to that of mTEC progenitors, has remained elusive. In this review, we describe distinct models that explain cTEC/mTEC lineage divergence from common bipotent progenitors. In particular, we summarize recent studies in mice providing evidence that mTECs, including the auto‐immune regulator+ subset, derive from progenitors initially endowed with phenotypic properties typically associated with the cTEC lineage. These observations support a novel “serial progression” model of TEC development, in which progenitors serially acquire cTEC lineage markers, prior to their commitment to the mTEC differentiation pathway. Gaining a better understanding of the phenotypic properties of early stages in TEC progenitor development should help in determining the mechanisms regulating cTEC/mTEC lineage development, and in strategies aimed at thymus reconstitution involving TEC therapy.

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.

CD117⁺ CD3⁻ CD56⁻ OX40Lhigh cells express IL-22 and display an LTi phenotype in human secondary lymphoid tissues.

Here, we identify cells within human adult secondary lymphoid tissues that are comparable in phenotype and location to the lymphoid tissue inducer (LTi) cells that persist in the adult mouse. Identified as CD117+CD3−CD56− cells, like murine LTi cells, they lack expression of many common lineage markers and express CD127, OX40L and TRANCE. These cells were detected at the interface between the B‐ and T‐ zones, as well as at the subcapsular sinus in LNs, the location where LTi cells reside in murine spleen and LNs. Furthermore, like murine LTi cells, these cells expressed high levels of IL‐22 and upregulated IL‐22 expression upon IL‐23 stimulation. Importantly, these cells were not an NK cell subset since they showed no expression of IFN‐γ and perforin. Interestingly, a subset of the CD117+CD3−CD56−OX40L+ population expressed NKp46, again similar to recent findings in mice. Finally, these cells supported memory CD4+ T‐cell survival in an OX40L‐dependent manner. Combined, these data indicate that the CD117+CD3−CD56−OX40L+ cells in human secondary lymphoid tissues are comparable in phenotype, location and function to the LTi cells that persist within adult murine secondary lymphoid tissues.

Generation of diversity in thymic epithelial cells

In the thymus, diverse populations of thymic epithelial cells (TECs), including cortical and medullary TECs and their subpopulations, have distinct roles in coordinating the development and repertoire selection of functionally competent and self-tolerant T cells. Here, we review the expanding diversity in TEC subpopulations in relation to their functions in T cell development and selection as well as their origins and development.

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 αβT‐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 mTEChi and mTEClo 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.

Mechanisms of Thymus Medulla Development and Function

The development of CD4(+) helper and CD8(+) cytotoxic T-cells expressing the αβ form of the T-cell receptor (αβTCR) 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 αβTCR 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 type 2 cytokine axis for thymus emigration

In the thymus, stromal microenvironments support a developmental program that generates mature T cells ready for thymic exit. The cellular and molecular specialization within thymic stromal cells that enables their regulation of specific stages of thymocyte development is poorly understood. Here, we show the thymic microenvironment expresses the type 2 IL-4R complex and is functionally responsive to its known ligands, IL-4 and IL-13. Absence of IL-4R&agr; limits thymocyte emigration, leading to an intrathymic accumulation of mature thymocytes within medullary perivascular spaces and reduced numbers of recent thymic emigrants. Thymus transplantation shows this requirement maps to IL-4R&agr; expression by stromal cells, and we provide evidence that it regulates thymic exit via a process distinct from S1P-mediated migration. Finally, we reveal a cellular mechanism by which IL-4+IL-13+ invariant NKT cells are necessary for IL-4R&agr; signaling that regulates thymic exit. Collectively, we define a new axis for thymic emigration involving stimulation of the thymic microenvironment via type 2 cytokines from innate T cells.

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

The thymus is a primary lymphoid tissue that supports the generation of αβT 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 αβT‐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.