554 VOLUME 15 NUMBER 6 JUNE 2014 NATURE IMMUNOLOGY The thymic microenvironment is unique in its capacity to promote the development of naive T cells. TECs are essential for this function as they control the attraction of blood-borne lymphoid precursors and subsequently facilitate their proliferation, differentiation and selec- tion to functionally competent T cells 1 . Based on their phenotype, anatomical localization and function, TECs can be distinguished into separate and functionally distinct subpopulations. Cortical (c)TECs initiate and control early stages of thymocyte development includ- ing the T cell antigen receptor (TCR)-mediated positive selection of cells that express a functional TCR 2,3 . In contrast, mTECs enforce the negative selection of thymocytes, control the formation of regulatory T cells, and influence the postselection maturation of naive T cells and their subsequent exit to the periphery 1,4 . TEC differentiation itself relies on signals mediated by developing T cells at distinct stages of their maturation, which provides an instructive illustration of cellular cross-talk in the thymus 5 . For example, engagement of the receptors RANK, CD40 or LTβ activates the noncanonical NF-κB pathway and consequently controls the differentiation of mTECs 6–11 as well as their capacity to express a broad range of tissue-restricted antigens (TRAs) 12 . These TRAs provide a largely comprehensive array of an individual’s antigens and thus present a molecular mirror of ‘immunological self’. Transcriptional regulation of the expression of TRAs is partly depend- ent on the transcriptional factor Aire, which is typically expressed by a subpopulation of mature mTECs 13,14 . A failure to correctly induce differentiation of mTECs and consequently expression of Aire compromises expression of TRAs and thus impacts the deletion of autoreactive T cells 15 . TGF-β is a widely expressed, highly conserved cytokine that acts on a large variety of cells 16 . TGF-β transmits its signals via binding to the type II receptor TGFβRII that consequently associates with the type I receptor TGFβRI and thus activates its serine/threonine kinase activity. In the thymus, TGF-β is expressed by cortical epithelial cells and immature thymocytes that release bioactive TGF-β as part of their programmed cell death 17,18 . A positive regulatory role for TGF-β signaling in development of intrathymic T cell has been proposed for CD8 + T cells, thymic regulatory T cells (tT reg cells) in very young mice and natural killer T cells 19 . In contrast, the importance of TGF-β sig- naling for TEC biology has not been sufficiently investigated though a loss of TGFβRII expression on TECs mitigates the physiological process of thymic senescence 20 . In this study, we provide evidence for a negative role of TGF-β signaling in mTECs, which limits their growth, differentiation and ultimately function. RESULTS TGF-b signaling controls the population expansion of mTECs cTECs and mTECs express mRNAs for both chains of the TGF-β receptor (ref. 20 and data not shown). To assess the role of TGF-β signaling for TEC growth, differentiation and function, we investi- gated mice with a targeted inactivation of Tgfbr2, the gene encoding the TGFβRII, in TECs. To delete TGFβRII specifically in TECs, we crossed mice with a loxP-flanked Tgfbr2 allele (Tgfbr2 fl/fl ) to mice expressing the Cre recombinase under the transcriptional control of the Foxn1 promoter 21 (FN1-Cre). We observed complete deletion of the targeted allele in TECs isolated from resultant homozygous mice (designated Tgfbr2 fl/fl FN1-Cre mice) as early as embryonic day 14 (E14) (Supplementary Fig. 1a). We first determined the frequency and phenotype of thymic epithelia isolated from mutant and control mice. We restricted this analysis to Tgfbr2 fl/fl FN1-Cre mice not older than 4 weeks of life 1 Immunology Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington, USA. 2 Laboratory of Pediatric Immunology, Department of Biomedicine, University of Basel, Basel, Switzerland. 3 Basel University’s Hospital, Basel, Switzerland. 4 Developmental Immunology, Department of Pediatrics, University of Oxford, Oxford, UK. 5 These authors contributed equally to this work. Correspondence should be addressed to S.F.Z. (sziegler@benaroyaresearch.org). Received 16 September 2013; accepted 11 March 2014; published online 13 April 2014; doi:10.1038/ni.2869 A regulatory role for TGF-b signaling in the establishment and function of the thymic medulla Mathias Hauri-Hohl 1 , Saulius Zuklys 2,3 , Georg A Holländer 2–5 & Steven F Ziegler 1,5 Medullary thymic epithelial cells (mTECs) are critical in establishing and maintaining the appropriate microenvironment for negative selection and maturation of immunocompetent T cells with a self-tolerant T cell antigen receptor repertoire. Cues that direct proliferation and maturation of mTECs are provided by members of the tumor necrosis factor (TNF) superfamily expressed on developing thymocytes. Here we demonstrate a negative role of the morphogen TGF- b in tempering these signals under physiological conditions, limiting both growth and function of the thymic medulla. Eliminating TGF-b signaling specifically in TECs or by pharmacological means increased the size of the mTEC compartment, enhanced negative selection and functional maturation of medullary thymocytes as well as the production of regulatory T cells, thus reducing the autoreactive potential of peripheral T cells. ARTICLES npg © 2014 Nature America, Inc. All rights reserved.