William E. Jenkinson

RANK signals from CD4+3− inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla

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.

Clonal analysis reveals a common progenitor for thymic cortical and medullary epithelium

The thymus provides an essential environment for the development of T cells from haemopoietic progenitors. This environment is separated into cortical and medullary regions, each containing functionally distinct epithelial populations that are important at successive stages of T-cell development and selection. However, the developmental origin and lineage relationships between cortical and medullary epithelial cell types remain controversial. Here we describe a clonal assay to investigate the developmental potential of single, individually selected, thymic epithelial progenitors (marked with enhanced yellow fluorescent protein) developing within the normal architecture of the thymus. Using this approach, we show that cortical and medullary epithelial cells share a common origin in bipotent precursors, providing definitive evidence that they have a single rather than dual germ layer origin during embryogenesis. Our findings resolve a long-standing issue in thymus development, and are important in relation to the development of cell-based strategies for thymus disorders and the possibility of restoring function of the atrophied adult thymus.

Low-level Hypermutation in T Cell–independent Germinal Centers Compared with High Mutation Rates Associated with T Cell–dependent Germinal Centers

Exceptionally germinal center formation can be induced without T cell help by polysaccharide-based antigens, but these germinal centers involute by massive B cell apoptosis at the time centrocyte selection starts. This study investigates whether B cells in germinal centers induced by the T cell–independent antigen (4-hydroxy-3-nitrophenyl)acetyl (NP) conjugated to Ficoll undergo hypermutation in their immunoglobulin V region genes. Positive controls are provided by comparing germinal centers at the same stage of development in carrier-primed mice immunized with a T cell–dependent antigen: NP protein conjugate. False positive results from background germinal centers and false negatives from non-B cells in germinal centers were avoided by transferring B cells with a transgenic B cell receptor into congenic controls not carrying the transgene. By 4 d after immunization, hypermutation was well advanced in the T cell–dependent germinal centers. By contrast, the mutation rate for T cell–independent germinal centers was low, but significantly higher than in NP-specific B cells from nonimmunized transgenic mice. Interestingly, a similar rate of mutation was seen in extrafollicular plasma cells at this stage. It is concluded that efficient activation of hypermutation depends on interaction with T cells, but some hypermutation may be induced without such signals, even outside germinal centers.

Differential Requirement for Mesenchyme in the Proliferation and Maturation of Thymic Epithelial Progenitors

Formation of a mature thymic epithelial microenvironment is an essential prerequisite for the generation of a functionally competent T cell pool. It is likely that recently identified thymic epithelial precursors undergo phases of proliferation and differentiation to generate mature cortical and medullary thymic microenvironments. The mechanisms regulating development of immature thymic epithelial cells are unknown. Here we provide evidence that expansion of embryonic thymic epithelium is regulated by the continued presence of mesenchyme. In particular, mesenchymal cells are shown to mediate thymic epithelial cell proliferation through their provision of fibroblast growth factors 7 and 10. In contrast, differentiation of immature thymic epithelial cells, including acquisition of markers of mature cortical and medullary epithelium, occurs in the absence of ongoing mesenchymal support. Collectively, our data define a role for mesenchymal cells in thymus development, and indicate distinct mechanisms regulate proliferation and differentiation of immature thymic epithelial cells. In addition, our findings may aid in studies aimed at developing strategies to enhance thymus reconstitution and functioning in clinical certain contexts where thymic epithelial cell function is perturbed.

The thymic medulla is required for Foxp3+ regulatory but not conventional CD4+ thymocyte development.

The thymic medulla and an intact mTEC compartment are needed for the development of nTreg cells and negative selection of conventional T cells but not their further maturation.

Checkpoints in the Development of Thymic Cortical Epithelial Cells

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.

Rank Signaling Links the Development of Invariant γδ T Cell Progenitors and Aire+ Medullary Epithelium

Summary 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 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.

Establishment and functioning of intrathymic microenvironments

Summary:  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.

Lymphotoxin Signals from Positively Selected Thymocytes Regulate the Terminal Differentiation of Medullary Thymic Epithelial Cells

The thymic medulla represents a key site for the induction of T cell tolerance. In particular, autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) provide a spectrum of tissue-restricted Ags that, through both direct presentation and cross-presentation by dendritic cells, purge the developing T cell repertoire of autoimmune specificities. Despite this role, the mechanisms of Aire+ mTEC development remain unclear, particularly those stages that occur post-Aire expression and represent mTEC terminal differentiation. In this study, in mouse thymus, we analyze late-stage mTEC development in relation to the timing and requirements for Aire and involucrin expression, the latter a marker of terminally differentiated epithelium including Hassall’s corpuscles. We show that Aire expression and terminal differentiation within the mTEC lineage are temporally separable events that are controlled by distinct mechanisms. We find that whereas mature thymocytes are not essential for Aire+ mTEC development, use of an inducible ZAP70 transgenic mouse line—in which positive selection can be temporally controlled—demonstrates that the emergence of involucrin+ mTECs critically depends upon the presence of mature single positive thymocytes. Finally, although initial formation of Aire+ mTECs depends upon RANK signaling, continued mTEC development to the involucrin+ stage maps to activation of the LTα–LTβR axis by mature thymocytes. Collectively, our results reveal further complexity in the mechanisms regulating thymus medulla development and highlight the role of distinct TNFRs in initial and terminal differentiation stages in mTECs.

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.