Eric J. Jenkinson

LYMPHOSTROMAL INTERACTIONS IN THYMIC DEVELOPMENT AND FUNCTION

The generation of a peripheral T-cell pool is essential for normal immune system function. CD4+ and CD8+ T cells are produced most efficiently in the thymus, which provides a complexity of discrete cellular microenvironments. Specialized stromal cells, that make up such microenvironments, influence each stage in the maturation programme of immature T-cell precursors. Progress has recently been made in elucidating events that regulate the development of intrathymic microenvironments, as well as mechanisms of thymocyte differentiation. It is becoming increasingly clear that the generation and maintenance of thymic environments that are capable of supporting efficient T-cell development, requires complex interplay between lymphoid and stromal compartments of the thymus.

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.

MHC CLASS II-POSITIVE EPITHELIUM AND MESENCHYME CELLS ARE BOTH REQUIRED FOR T-CELL DEVELOPMENT IN THE THYMUS

T LYMPHOCYTES are produced in the thymus from precursors originating in the haemopoietic tissues. On entering the thymus, they undergo a programme of proliferation, T-cell receptor (TCR) gene rearrangement, differentiation and repertoire selection1. Although the thymus provides a unique environment for these events, the role of the thymic stroma in regulating specific developmental stages is not well understood2. We therefore devised an in vitro system to study the role of individual thymic stromal components in T-cell development. We report here that the development of TCR–CD4–CD8– T-cell precursors into TCR+ cells expressing CD4 and/or CDS requires the presence of both major histocom-patibility complex class II+ epithelial cells and fetal mesenchyme. The requirement for mesenchymal support can be mapped to the initial stages of intrathymic development because the later stages of maturation, from double-positive CD4+CD8+ thymocytes into single-positive CD4+ or CD8+ cells, can be supported by epithelial cells alone. We also show that the requirement for mesenchymal cells can be met by cells of the fib rob last line 3T3 (but not by supernatants from these cells). To our knowledge, these findings provide the first direct evidence that mesenchymal as well as epithelial cells are involved in T-cell development, and suggest that their involvement is stage-specific and likely to be dependent on short-range or contact-mediated interactions.

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.

Progression through key stages of haemopoiesis is dependent on distinct threshold levels of c-Myb.

The c‐Myb transcription factor is expressed in immature haemopoietic cells and at key stages during differentiation. Loss of the c‐myb gene results in embryonic lethality because mature blood cells fail to develop, although commitment to definitive haemopoiesis occurs. We have generated a knockdown allele of c‐myb, expressing low levels of the protein, which has enabled us to investigate further the involvement of c‐Myb in haemopoiesis. Low levels of c‐Myb are sufficient to allow progenitor expansion but, importantly, we show that progression of progenitors towards terminal differentiation is significantly altered. Suboptimal levels of c‐Myb favour differentiation of macrophage and megakaryocytes, while higher levels seem to be particularly important in the control of erythropoiesis and lymphopoiesis. We provide evidence that the transition from the CFU‐E to erythroblasts is critically dependent on c‐Myb levels. During thymopoiesis, c‐Myb appears to regulate immature cell numbers and differentiation prior to expression of CD4 and CD8. Overall, our results point to a complex involvement of c‐Myb in the regulation of proliferation and commitment within the haemopoietic hierarchy.

Generating intrathymic microenvironments to establish T-cell tolerance

αβ T cells pass through a series of lymphoid tissue stromal microenvironments to acquire self tolerance and functional competence. In the thymus, positive selection of the developing T-cell receptor repertoire occurs in the cortex, whereas events in the medulla purge the system of self reactivity. T cells that survive are exported to secondary lymphoid organs where they direct first primary and then memory immune responses. This Review focuses on the microenvironments that nurture T-cell development rather than on T cells themselves. We summarize current knowledge on the formation of thymic epithelial-cell microenvironments, and highlight similarities between the environments that produce T cells and those that select and maintain them during immune responses.

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.

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.

Thymic epithelial cells provide Wnt signals to developing thymocytes

Interactions with thymic stromal cells are known to be critical for the development of T cells from progenitors entering the thymus, yet the molecular mechanisms of stromal cell function remain poorly understood. Accumulating evidence has highlighted the importance of β‐catenin‐mediated activation of T cell factor (TCF)/lymphoid enhancer factor (LEF) transcription during thymocyte development. As regulation of this signaling pathway is controlled by binding of soluble Wnt proteins to cell surface Frizzled (Fz) receptors, we studied components of Wnt/Fz‐mediated signaling in thecontext of stromal cell regulation of thymocyte development. We show that mRNA for a variety of Wnt family members, notably Wnt‐4, Wnt‐7a and 7b, and Wnt‐10a and 10b, are expressed by thymic epithelium rather then by thymocytes, while thymocytes demonstrate a developmentally regulated pattern of Fz receptor expression. Collectively these findings suggest (1) a functional role for Wnt‐producing thymic epithelium in determining TCF/LEF‐mediated transcriptional regulation in Fz‐bearing thymocytes, and (2) a role for defined Wnt‐Fz interactions at successive stages of thymocyte maturation. In support of this we show that separation of thymocytes from Wnt‐producing epithelial cells and the thymic microenvironment, triggers β‐catenin phosphorylation and degradation in thymocytes. Thus, sustained exposure to Wnt in the context of an intact stromal microenvironment is necessary for stabilization of β‐catenin‐mediated signaling in thymocytes.