Conrad C. Bleul

Thymic Medullary Epithelial Cell Differentiation, Thymocyte Emigration, and the Control of Autoimmunity Require Lympho–Epithelial Cross Talk via LTβR

Thymocytes depend on the interaction with thymic epithelial cells for the generation of a diverse, nonautoreactive T cell repertoire. In turn, thymic epithelial cells acquire their three-dimensional cellular organization via instructive signals from developing thymocytes. The nature of these signals has been elusive so far. We show that thymocytes and medullary epithelial cells (MECs) communicate via the lymphotoxin β receptor (LTβR) signaling axis. Normal differentiation of thymic MECs requires LTβR ligand on thymocytes and LTβR together with nuclear factor–κB-inducing kinase (Nik) in thymic epithelial cells. Impaired lympho–epithelial cross talk in the absence of the LTβR causes aberrant differentiation and reduced numbers of thymic MECs, leads to the retention of mature T lymphocytes, and is associated with autoimmune phenomena, suggesting an unexpected role for LTβR signaling in central tolerance induction.

Formation of a functional thymus initiated by a postnatal epithelial progenitor cell

The thymus is essential for the generation of self-tolerant effector and regulatory T cells. Intrathymic T-cell development requires an intact stromal microenvironment, of which thymic epithelial cells (TECs) constitute a major part. For instance, cell-autonomous genetic defects of forkhead box N1 (Foxn1) and autoimmune regulator (Aire) in thymic epithelial cells cause primary immunodeficiency and autoimmunity, respectively. During development, the thymic epithelial rudiment gives rise to two major compartments, the cortex and medulla. Cortical TECs positively select T cells, whereas medullary TECs are involved in negative selection of potentially autoreactive T cells. It has long been unclear whether these two morphologically and functionally distinct types of epithelial cells arise from a common bi-potent progenitor cell and whether such progenitors are still present in the postnatal period. Here, using in vivo cell lineage analysis in mice, we demonstrate the presence of a common progenitor of cortical and medullary TECs after birth. To probe the function of postnatal progenitors, a conditional mutant allele of Foxn1 was reverted to wild-type function in single epithelial cells in vivo. This led to the formation of small thymic lobules containing both cortical and medullary areas that supported normal thymopoiesis. Thus, single epithelial progenitor cells can give rise to a complete and functional thymic microenvironment, suggesting that cell-based therapies could be developed for thymus disorders.

A multipotent precursor in the thymus maps to the branching point of the T versus B lineage decision

Hematopoietic precursors continuously colonize the thymus where they give rise mainly to T cells, but also to B and dendritic cells. The lineage relationship between these three cell types is unclear, and it remains to be determined if precursors in the thymus are multipotent, oligopotent, or lineage restricted. Resolution of this question necessitates the determination of the clonal differentiation potential of the most immature precursors in the thymus. Using a CC chemokine receptor 9–enhanced green fluorescent protein knock-in allele like a surface marker of unknown function, we identify a multipotent precursor present in bone marrow, blood, and thymus. Single cells of this precursor give rise to T, B, and dendritic cells. A more differentiated stage of this multipotent precursor in the thymus has lost the capacity to generate B but not T, dendritic, and myeloid cells. Thus, the newly identified precursor maps to the branching point of the T versus B lineage decision in the hematopoietic lineage hierarchy.

BMP Signaling Is Required for Normal Thymus Development

The microenvironment of the thymus fosters the generation of a diverse and self-tolerant T cell repertoire from a pool of essentially random specificities. Epithelial as well as mesenchymal cells contribute to the thymic stroma, but little is known about the factors that allow for communication between the two cells types that shape the thymic microenvironment. In this study, we investigated the role of bone morphogenetic protein (BMP) signaling in thymus development. Transgenic expression of the BMP antagonist Noggin in thymic epithelial cells under the control of a Foxn1 promoter in the mouse leads to dysplastic thymic lobes of drastically reduced size that are ectopically located in the neck at the level of the hyoid bone. Interestingly, the small number of thymocytes in these thymic lobes develops with normal kinetics and shows a wild-type phenotype. Organ initiation of the embryonic thymic anlage in these Noggin transgenic mice occurs as in wild-type mice, but the tight temporal and spatial regulation of BMP4 expression is abrogated in subsequent differentiation stages. We show that transgenic Noggin blocks BMP signaling in epithelial as well as mesenchymal cells of the thymic anlage. Our data demonstrate that BMP signaling is crucial for thymus development and that it is the thymic stroma rather than developing thymocytes that depends on BMP signals.

The evolutionary history of lymphoid organs

Lymphoid organs are important regulators of lymphocyte development and immune responses. During vertebrate evolution, primary lymphoid organs appeared earlier than secondary lymphoid organs. Among the sites of primary lymphopoiesis during evolution and ontogeny, those for B cell differentiation have differed considerably, although they often have had myelolymphatic characteristics. In contrast, only a single site for T cell differentiation has occurred, exclusively the thymus. Based on those observations and the known features of variable-diversity-joining gene recombination, we propose a model for the successive specification of different lymphocyte lineages during vertebrate evolution. According to our model, T cells were the first lymphocytes to acquire variable-diversity-joining–type receptors, and the thymus was the first lymphoid organ to evolve in vertebrates to deal with potentially autoreactive, somatically diversified T cell receptors.

Homing of immature thymocytes to the subcapsular microenvironment within the thymus is not an absolute requirement for T cell development

T cell development is thought to occur in distinct microenvironments within the thymus. Namely, the subcapsular zone, the cortex and the medulla have been described to support expansion of the immature thymocyte pool, positive selection of useful specificities and elimination of potentially self‐reactive specificities, respectively. Consistent with this model, thymocytes show a highly ordered migration pattern and move into these niches in the expected sequence. Here we show that the chemokine receptor CCR9 plays a nonredundant role in the homing of immature thymocytes to the subcapsular zone. In CCR9‐deficient mice, T cells in early stages of development do not accumulate in their physiological microenvironment underneath the thymic capsule and are instead homogeneously distributed across the thymic cortex. Remarkably, this abnormality does not result in a detectable defect in T cell development in CCR9‐deficient mice, suggesting that the transit of immature thymocytes through the subcapsular microenvironment is not an absolute requirement for proper T cell development.

The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development

T cell development in the thymus depends on continuous colonization by hematopoietic precursors. Several distinct T cell precursors have been identified, but whether one or several independent precursor cell types maintain thymopoiesis is unclear. We have used thymus transplantation and an inducible lineage-tracing system to identify the intrathymic precursor cells among previously described thymus-homing progenitors that give rise to the T cell lineage in the thymus. Extrathymic precursors were not investigated in these studies. Both approaches show that the stream of T cell lineage precursor cells, when entering the thymus, selectively passes through the early T lineage precursor (ETP) stage. Immigrating precursor cells do not exhibit characteristics of double-negative (DN) 1c, DN1d, or DN1e stages, or of populations containing the common lymphoid precursor 2 (CLP-2) or the thymic equivalent of circulating T cell progenitors (CTPs). It remains possible that an unknown hematopoietic precursor cell or previously described extrathymic precursors with a CLP, CLP-2, or CTP phenotype feed into T cell development by circumventing known intrathymic T cell lineage progenitor cells. However, it is clear that of the known intrathymic precursors, only the ETP population contributes significant numbers of T lineage precursors to T cell development.

Bone Marrow-Derived Hemopoietic Precursors Commit to the T Cell Lineage Only after Arrival in the Thymic Microenvironment

T lymphocytes develop in the thymus from hemopoietic precursors that commit to the T cell lineage under the influence of Notch signals. In this study, we show by single cell analyses that the most immature hemopoietic precursors in the adult mouse thymus are uncommitted and specify to the T cell lineage only after their arrival in the thymus. These precursors express high levels of surface Notch receptors and rapidly lose B cell potential upon the provision of Notch signals. Using a novel culture system with complexed, soluble Notch ligands that allows the titration of T cell lineage commitment, we find that these precursors are highly sensitive to both Delta and Jagged ligands. In contrast, their phenotypical and functional counterparts in the bone marrow are resistant to Notch signals that efficiently induce T cell lineage commitment in thymic precursors. Mechanistically, this is not due to differences in receptor expression, because early T lineage precursors, bone marrow lineage marker-negative, Sca-1-positive, c-Kit-positive and common lymphoid progenitor cells, express comparable amounts of surface Notch receptors. Our data demonstrate that the sensitivity to Notch-mediated T lineage commitment is stage-dependent and argue against the bone marrow as the site of T cell lineage commitment.

Genetic dissection of thymus development in mouse and zebrafish

Summary:  Lymphoid organs represent a specialized microenvironment for interaction of stromal and lymphoid cells. In primary lymphoid organs, these interactions are required to establish a self‐tolerant repertoire of lymphocytes. While detailed information is available about the genes that control lymphocyte differentiation, little is known about the genes that direct the establishment and differentiation of principal components of such microenvironments. Here, we discuss genetic studies addressing the role of thymic epithelial cells (TECs) during thymopoiesis. We have identifed an evolutionarily conserved key regulator of TEC differentiation, Foxn1, that is required for the immigration of prothymocytes into the thymic primordium. Because Foxn1 specifies the prospective endodermal domain that gives rise to thymic epithelial cells, it can be used to identify the evolutionary origins of this specialized cell type. In the course of these studies, we have found that early steps of thymus development in zebrafish are very similar to those in mice. Subsequently, we have used chemical mutagenesis to derive zebrafish lines with aberrant thymus development. Strengths and weaknesses of mouse and zebrafish models are largely complementary such that genetic analysis of mouse and zebrafish mutants may lead to a better understanding of thymus development.

Laser capture microdissection-based expression profiling identifies PD1-ligand as a target of the nude locus gene product

T cell development requires the interaction of developing thymocytes with thymic epithelial cells. Thymic epithelial cells acquire their unique phenotype under the control of the winged‐helix transcription factor Whn, which is lacking in the nude mouse. Whn‐dependent genes may therefore be important regulators of lympho‐epithelial interactions. To identify Whn target genes we isolated RNA populations of wild‐type and nude thymic anlagen from embryonic day 12.5 embryos by laser capture microdissection and compared them by gene expression profiling on microarrays representing 22,000 transcripts. All cDNA with expression differences confirmed by quantitative RT‐PCR using RNA from individual anlagen and by in situ hybridization were found to be present in wild‐type and absent in nude samples. Three of eight confirmed transcripts were of hematopoietic origin; these transcripts emanate from hematopoietic precursors which have just entered the thymic anlage. Five transcripts were of epithelial origin; one of these corresponds to the recently identified PD‐1 ligand (PD‐L1), the receptor of which is known to modulate positive selection and to play a role in the control of autoimmunity, and the remaining transcripts code for novel genes. The presented results support our prediction that this systematic approach by gene expression profiling yields regulators of thymopoiesis.