Cell competition is an emerging principle underlying selection for cellular fitness during development and disease. Competition may be relevant for cancer, but an experimental link between defects in competition and tumorigenesis is elusive. In the thymus, T lymphocytes develop from precursors that are constantly replaced by bone-marrow-derived progenitors. Here we show that in mice this turnover is regulated by natural cell competition between 'young' bone-marrow-derived and 'old' thymus-resident progenitors that, although genetically identical, execute differential gene expression programs. Disruption of cell competition leads to progenitor self-renewal, upregulation of Hmga1, transformation, and T-cell acute lymphoblastic leukaemia (T-ALL) resembling the human disease in pathology, genomic lesions, leukaemia-associated transcripts, and activating mutations in Notch1. Hence, cell competition is a tumour suppressor mechanism in the thymus. Failure to select fit progenitors through cell competition may explain leukaemia in X-linked severe combined immune deficiency patients who showed thymus-autonomous T-cell development after therapy with gene-corrected autologous progenitors.

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T cell development in the thymus depends on continuous colonization by hematopoietic precursors. Several distinct T cell precursors have been identifi ed, 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.

The forkhead box N1 (Foxn1) protein is the key regulator of thymic epithelial cell (TEC) development, yet how Foxn1 functions remains largely unknown. All mature TECs arise from Foxn1-expressing progenitors/immature TECs and it is widely assumed that TECs as a whole are defined by Foxn1 expression. However, data on the Foxn1 protein are virtually lacking. In this study, we developed novel tools to visualize Foxn1 protein expression at single-cell resolution. We generated Foxn1 knock-in mice expressing a C-terminal hemagglutinin-tagged Foxn1 protein, and a cytometry-grade monoclonal anti-Foxn1 Ab. We evaluated Foxn1 expression patterns in TEC subsets and its dynamics during normal thymus development, aging, injury, and regeneration. Upon challenges, upregulation of Foxn1 was a common feature of thymus regeneration, but the timing of Foxn1 expression changed and the responding TEC subsets depended on the type of treatment. Whereas dexamethasone and recombinant human fibroblast growth factor 7 promoted expansion of Foxn1+Ly51+CD80− TECs, castration led to expansion of Foxn1+Ly51−CD80+ TECs. Collectively, Foxn1 expression is highly heterogeneous in the normal thymus, with large fractions of Foxn1low or Foxn1− TECs accumulating with age. Furthermore, Foxn1 expression is responsive to perturbations.

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.

Thymic medullary epithelial cells (mTECs) play a major role in central tolerance induction by expressing tissue-specific Ags (TSAs). The expression of a subset of TSAs in mTECs is under the control of Aire (autoimmune regulator). Humans defective for AIRE develop a syndrome characterized by autoimmune disease in several endocrine glands. Aire has been proposed to be regulated by lymphotoxin ␤ receptor (Lt␤r) signaling and there is evidence that, additionally, Aire-independent transcripts may be regulated by this pathway. Given the potential clinical importance of Aire regulation in mTECs for the control of autoimmunity, we investigated the relation between Lt␤r signaling and TSA expression by whole genome transcriptome analysis. In this study, we show that the absence of Lt␤r has no effect on the expression of Aire and Aire-dependent TSAs. Also, the lack of Lt␤r signaling does not disturb regulatory T cells or the distribution of dendritic cells in the thymus. However, mTECs in Lt␤r-deficient mice show an aberrant distribution within the thymic medulla with disruption of their three-dimensional architecture. This is predicted to impair the interaction between mTECs and thymocytes as shown by the reduced surface uptake of MHCII by mature thymocytes in Lt␤r-deficient mice. We propose that the physiological medullary architecture ensures negative-selection by supporting lympho-epithelial interaction through a large epithelial cell surface distributed evenly across the medulla.

A novel pre-TCRα (pTα) reporter mouse reveals that expression of pTα is confined to the T lineage and does not occur on prethymic progenitors.

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