Simona W. Rossi

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.

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.

Redefining epithelial progenitor potential in the developing thymus

Cortical and medullary epithelium represent specialised cell types that play key roles in thymocyte development, including positive and negative selection of the T cell repertoire. While recent evidence shows that these epithelial lineages share a common embryonic origin, the phenotype and possible persistence of such progenitor cells in the thymus at later stages of development remain controversial. Through use of a panel of reagents including the putative progenitor marker Mts24, we set out to redefine the stages in the development of thymic epithelium. In the early embryonic day (E)12 thymus anlagen we find that almost all epithelial cells are uniformly positive for Mts24 expression. In addition, while the thymus at later stages of development was found to contain distinct Mts24+ and Mts24– epithelial subsets, thymus grafting experiments show that both Mts24+ and Mts24– epithelial subsets share the ability to form organised cortical and medullary thymic microenvironments that support T cell development, a function shown previously to be lost in the Mts24– cells by E15 when lower cell doses were used. Our data help to clarify stages in thymic epithelial development and provide important information in relation to currently used markers of epithelial progenitors.

AIRE's CARD Revealed, a New Structure for Central Tolerance Provokes Transcriptional Plasticity

Developing T cells encounter peripheral self-antigens in the thymus in order to delete autoreactive clones. It is now known that the autoimmune regulator protein (AIRE), which is expressed in thymic medullary epithelial cells, plays a key role in regulating the thymic transcription of these peripheral tissue-specific antigens. Mutations in the AIRE gene are associated with a severe multiorgan autoimmune syndrome (APECED), and autoimmune reactivities are manifest in AIRE-deficient mice. Functional AIRE protein is expressed as distinct nuclear puncta, although no structural basis existed to explain their relevance to disease. In addressing the cell biologic basis for APECED, we made the unexpected discovery that an AIRE mutation hot spot lies in a caspase recruitment domain. Combined homology modeling and in vitro data now show how APECED mutations influence the activity of this transcriptional regulator. We also provide novel in vivo evidence for AIREs association with a global transcription cofactor, which may underlie AIREs focal, genome-wide, alteration of the transcriptome.

Heterogeneity of lymphoid tissue inducer cell populations present in embryonic and adult mouse lymphoid tissues

Lymphoid tissue inducer (LTi) cells have a well established role in secondary lymphoid tissue development. Here, we report on the heterogeneity of LTi cells based on their CD4 and chemokine receptor expression. The CD4− LTi‐cell population has a similar phenotype to the CD4+ population, with similar chemokine‐receptor‐expressing subsets. In both embryonic and adult spleen the CD4− LTi‐cell population is comparable as a proportion of total splenocytes to its CD4+ counterpart. In contrast, different proportions of CD4+ and CD4− LTi cells are found in different lymph nodes. Both CD4+ and CD4− LTi cells share the anatomical location and are associated with vascular cell adhesion molecule‐1‐positive stromal cells in spleen and lymph nodes. The numbers of both CD4+ and CD4− LTi cells in adult spleen are augmented in the presence of B cells. With the exception of CD4, there is a strong correlation coefficient (0·89) for gene expression between the two populations. Polymerase chain reaction analysis of individual CD4+ and CD4− LTi cells shows that a similar proportion in embryonic and adult spleen co‐expressed both CXCR5 and CCR7 or CXCR5 alone: 84·6% for adult CD4+ and 87·6% for adult CD4−; 95·3% for embryonic CD4+ and 91·5% for embryonic CD4−. Consistently fewer CCR7 single‐positive cells were found in the CD4+ and CD4− fractions in the embryo.

The role of lymphoid tissue inducer cells in splenic white pulp development

CD4+CD3– lymphoid tissue inducer (LTi) cells are crucial for the development and organisation of lymph nodes and gut associated lymphoid tissues. In this report, we characterise their appearance in the developing spleen and highlight their importance in relation to the development of splenic T cell zones. LTi cells were detected in embryonic spleen from embryonic day 13, although their progenitors were present at embryonic day 12. These cells clustered initially around splenic blood vessels in a lymphotoxin (LT)‐independent manner, but up‐regulation of VCAM‐1 expression on stromal cells associated with the blood vessels was LT dependent. After birth, T cell colonisation of these clusters to form nascent white pulp areas was also LT dependent. Transfer experiments reconstituting RAG–/– mice with either WT or LTα–/– splenocytes demonstrated that lymphocyte expression of LT was not essential for the organisation of a discrete CD3+ T cell zone with localised podoplanin and CCL21 expression. Our studies indicate that a combination of LT signals from LTi cells and LT‐independent signals from lymphocytes is sufficient for expression of podoplanin and CCL21 on splenic T cell zone stroma and subsequent T cell organisation.

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.

Development of functional thymic epithelial cells occurs independently of lymphostromal interactions

The thymus provides a specialised microenvironment for the development of T-cell precursors. This developmental programme depends upon interactions with stromal cells such as thymic epithelial cells, which provide signals for proliferation, survival and differentiation. In turn, it has been proposed that development of thymic epithelial cells themselves is regulated by signals produced by developing thymocytes. Evidence in support of this symbiotic relationship, termed thymic crosstalk, comes from studies analysing the thymus of adult mice harbouring blocks at specific stages of thymocyte development, where it is difficult to separate mechanisms regulating the initial development of thymic epithelial cells from those regulating their maintenance. To distinguish between these processes, we have analysed the initial developmental programme of thymic epithelial cells within the embryonic thymus, in either the presence or absence of normal T-cell development. We show that keratin 5+8+ precursor epithelial cells present in the early thymic rudiment differentiate into discrete cortical and medullary epithelial subsets displaying normal gene expression profiles, and acquire functional competence, independently of signals from T-cell precursors. Thus, our findings redefine current models of thymus development and argue against a role for thymocyte-epithelial cell crosstalk in the development of thymic epithelial progenitors.

Chemokine receptor expression defines heterogeneity in the earliest thymic migrants

Chemokine signaling has been implicated in directing colonization of the fetal thymus by hematopoietic precursors. However, the patterns of expression of the chemokine receptors responsible for directing thymic colonization by the earliest thymic migrants remain unknown. We have identified heterogeneity within the earliest thymus seeding cells based on chemokine receptor expression. By analyzing the first wave of progenitors to colonize the thymus at E12 of gestation, we show that multiple chemokine receptors are expressed by T‐lymphoid precursors present within perithymic mesenchyme, while expression of chemokine ligands is limited to CCL21, CCL25 and CXCL12, which are located in distinct epithelial and mesenchymal compartments of the thymic/parathyroid anlagen. Collectively, these results identify multiple populations of T‐lymphoid precursors colonizing the fetal thymus and provide evidence for several potential pathways mediating migration of precursors into the embryonic thymus.