Thymic epithelial cells (TECs) help orchestrate thymopoiesis, and TEC differentiation relies on bidirectional interactions with thymocytes. Although the molecular mediators that stimulate medullary thymic epithelial cell (mTEC) maturation are partially elucidated, the signals that regulate cortical thymic epithelial cell (cTEC) homeostasis remain elusive. Using IL-7 reporter mice, we show that TECs coexpressing high levels of IL-7 (Il7YFP+ TECs) reside within a subset of CD205+Ly51+CD40low cTECs that coexpresses Dll4, Ccl25, Ccrl1, Ctsl, Psmb11, and Prss16 and segregates from CD80+CD40high mTECs expressing Tnfrsf11a, Ctss, and Aire. As the frequency of Il7YFP+ TECs gradually declines as mTEC development unfolds, we explored the relationship between Il7YFP+ TECs and mTECs. In thymic organotypic cultures, the thymocyte-induced reduction in Il7YFP+ TECs dissociates from the receptor activator of NF-κB–mediated differentiation of CD80+ mTECs. Still, Il7YFP+ TECs can generate some CD80+ mTECs in a stepwise differentiation process via YFP−Ly51lowCD80low intermediates. Il7YFP+ TECs are sustained in Rag2−/− mice, even following in vivo anti-CD3ε treatment that mimics the process of pre-TCR β-selection of thymocytes to the double positive (DP) stage. Using Marilyn-Rag2−/− TCR transgenic, we find that positive selection into the CD4 lineage moderately reduces the frequency of Il7YFP+ TECs, whereas negative selection provokes a striking loss of Il7YFP+ TECs. These results imply that the strength of MHC/peptide–TCR interactions between TECs and thymocytes during selection constitutes a novel rheostat that controls the maintenance of IL-7–expressing cTECs.

Key points:• TEC-intrinsic ablation of p53 predominantly affects medullary TECs, altering their RANK-driven differentiation and transcriptome.• Loss of p53 in TECs couples disrupted thymopoiesis to altered T-cell homeostasis and tolerance Thymic epithelial cells (TECs) provide crucial microenvironments for T-cell development and tolerance induction. As the regular function of the thymus declines with age, it is of fundamental and clinical relevance to decipher new determinants that control TEC homeostasis in vivo. Beyond its recognized tumor suppressive function, p53 controls several immunoregulatory pathways. To study the cell-autonomous role of p53 in thymic epithelium functioning, we developed and analyzed mice with conditional inactivation of Trp53 in TECs (p53cKO). We report that loss of p53 primarily disrupts the integrity of medullary TEC (mTEC) niche, a defect that spreads to the adult cortical TEC compartment. Mechanistically, we found that p53 controls specific and broad programs of mTEC differentiation. Apart from restraining the expression and responsiveness of the receptor activator of NF-kB (RANK), which is central for mTEC differentiation, deficiency of p53 in TECs altered multiple functional modules of the mTEC transcriptome, including tissuerestricted antigen expression. As a result, p53cKO mice presented premature defects in mTEC-dependent regulatory T-cell differentiation and thymocyte maturation, which progressed to a failure in regular and regenerative thymopoiesis and peripheral Tcell homeostasis in the adulthood. Lastly, peripheral signs of altered immunological tolerance unfold in mutant mice and in immunodeficient mice that received p53cKO-derived thymocytes. Our findings position p53 as a novel molecular determinant of thymic epithelium function throughout life.Version: Postprint (identical content as published paper) This is a self-archived document from i3S -Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00 IntroductionWithin the thymus, thymic epithelial cells (TECs) orchestrate the development of functionally diverse and self-tolerant T cells. 1 Importantly, impaired TEC functions arise with aging, cytoablative regimens and infection, which compromise T-cell responses to pathogens, and vaccination in the elderly, and patients undergoing bone marrow transplantation (BMT) or chemotherapy. Equally, failures in TEC-mediated tolerance induction lead to autoimmunity. 2 Hence, the identification of novel regulators of TEC homeostasis is crucial to comprehend the foundations of immunity and to intervene medically in disorders linked to a dysfunctional thymus.Cortical TECs (cTECs) and medullary TECs (mTECs) define 2 functionally distinct microenvironments, which differentiate from bipotent TEC progenitors. 1 Whereas cTECs drive T-cell lineage specification and positive selection, mTECs promote the maturation of positively selected thymocytes, regulatory T-c...

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Cortical (cTEC) and medullary (mTEC) thymic epithelial cells establish key microenvironments for T-cell differentiation and arise from thymic epithelial cell progenitors (TEP). However, the nature of TEPs and the mechanism controlling their stemness in the postnatal thymus remain poorly defined. Using TEC clonogenic assays as a surrogate to survey TEP activity, we found that a fraction of cTECs generates specialized clonal-derived colonies, which contain cells with sustained colony-forming capacity (ClonoTECs). These ClonoTECs are EpCAM+MHCII-Foxn1lo cells that lack traits of mature cTECs or mTECs but co-express stem-cell markers, including CD24 and Sca-1. Supportive of their progenitor identity, ClonoTECs reintegrate within native thymic microenvironments and generate cTECs or mTECs in vivo. Strikingly, the frequency of cTECs with the potential to generate ClonoTECs wanes between the postnatal and young adult immunocompetent thymus, but it is sustained in alymphoid Rag2-/-Il2rg-/-counterparts. Conversely, transplantation of wild-type bone marrow hematopoietic progenitors into Rag2-/-Il2rg-/-mice and consequent restoration of thymocyte-mediated TEC differentiation diminishes the frequency of colony-forming units within cTECs. Our findings provide evidence that the cortical epithelium contains a reservoir of epithelial progenitors whose abundance is dynamically controlled by continual interactions with developing thymocytes across lifespan.

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