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Autoimmune regulator+ (Aire) medullary thymic epithelial cells (mTECs) play a critical role in tolerance induction. Several studies demonstrated that Aire+mTECs differentiate further into Post-Aire cells. Yet, the identification of terminal stages of mTEC maturation depends on unique fate-mapping mouse models. Herein, we resolve this limitation by segmenting the mTEChi(MHCIIhiCD80hi) compartment into mTECA/hi (CD24−Sca1−), mTECB/hi (CD24+Sca1−), and mTECC/hi (CD24+Sca1+). While mTECA/hi included mostly Aire-expressing cells, mTECB/hi contained Aire+ and Aire− cells and mTECC/hi were mainly composed of cells lacking Aire. The differential expression pattern of Aire led us to investigate the precursor-product relationship between these subsets. Strikingly, transcriptomic analysis of mTECA/hi, mTECB/hi, and mTECC/hi sequentially mirrored the specific genetic program of Early-, Late- and Post-Aire mTECs. Corroborating their Post-Aire nature, mTECC/hi downregulated the expression of tissue-restricted antigens, acquired traits of differentiated keratinocytes, and were absent in Aire-deficient mice. Collectively, our findings reveal a new and simple blueprint to survey late stages of mTEC differentiation.

Despite the essential role of thymic epithelial cells (TEC) in T cell development, the signals regulating TEC differentiation and homeostasis remain incompletely understood. In this study, we show a key in vivo role for the vitamin A metabolite, retinoic acid (RA), in TEC homeostasis. In the absence of RA signaling in TEC, cortical TEC (cTEC) and CD80loMHC class IIlo medullary TEC displayed subset-specific alterations in gene expression, which in cTEC included genes involved in epithelial proliferation, development, and differentiation. Mice whose TEC were unable to respond to RA showed increased cTEC proliferation, an accumulation of stem cell Ag-1hi cTEC, and, in early life, a decrease in medullary TEC numbers. These alterations resulted in reduced thymic cellularity in early life, a reduction in CD4 single-positive and CD8 single-positive numbers in both young and adult mice, and enhanced peripheral CD8+ T cell survival upon TCR stimulation. Collectively, our results identify RA as a regulator of TEC homeostasis that is essential for TEC function and normal thymopoiesis.

Despite the key role of primary and secondary lymphoid organ stroma in immunity, our understanding of the heterogeneity and ontogeny of these cells remains limited. Here, we identify a functionally distinct subset of BP3-PDPN+PDGFRβ+/α+CD34+ stromal adventitial cells in both lymph nodes (LNs) and thymus that is located within the vascular niche surrounding PDPN-PDGFRβ+/α-Esam-1+ITGA7+ pericytes. CD34+ adventitial cells developed in late embryonic thymus and in postnatal LNs and in the thymus originated, along with pericytes, from a common anlage-seeding progenitor population. Using lymphoid organ re-aggregate grafts, we demonstrate that adult CD34+ adventitial cells are capable of differentiating into multiple lymphoid stroma-like subsets including pericyte-, FRC-, MRC-, and FDC-like cells, the development of which was lymphoid environment-dependent. These findings extend the current understanding of lymphoid mesenchymal cell heterogeneity and highlight a role of the CD34+ adventitia as a potential ubiquitous source of lymphoid stromal precursors in postnatal tissues.

Despite the key role of primary and secondary lymphoid organ stroma in immunity, our understanding of the heterogeneity and ontogeny of these cells remains limited. Here, we identify a functionally distinct subset of BP3(-)PDPN(+)PDGFR beta(+)/alpha(+)CD34(+) stromal adventitial cells in both lymph nodes (LNs) and thymus that is located within the vascular niche surrounding PDPN(-)PDGFR beta(+)/alpha(-)Esam-1(+)ITGA7(+) pericytes. CD34(+) adventitial cells developed in late embryonic thymus and in postnatal LNs and in the thymus originated, along with pericytes, from a common anlage-seeding progenitor population. Using lymphoid organ re-aggregate grafts, we demonstrate that adult CD34(+) adventitial cells are capable of differentiating into multiple lymphoid stroma-like subsets including pericyte- ,FRC-, MRC-, and FDC-like cells, the development of which was lymphoid environment-dependent. These findings extend the current understanding of lymphoid mesenchymal cell heterogeneity and highlight a role of the CD34(+) adventitia as a potential ubiquitous source of lymphoid stromal precursors in postnatal tissues.

The Vitamin A derivative retinoic acid (RA) works as a ligand for a family of nuclearRA receptors (RARα, RARβ and RARγ) which form heterodimers with retinoid Xreceptors (RXR). These complexes function as ligand-activated transcription factors,recognizing specific RA responsive elements in the regulatory regions of targetgenes. RA has been reported to play a direct role in regulating multiple aspects of peripheralT cell responses1, but whether endogenous RA signalling occurs in developingthymocytes and the potential impact of such signals in regulating T cell developmentremains unclear. To address this question, we have crossed dnRARα mice, that carry a dominantnegative(dn) RARα transgene behind a floxed transcriptional STOP cassette, withCD4Cre mice. In the resulting dnRARα-CD4Cre+ mice, CD4-promotor driven Creexpressionremoves the STOP cassette and leads to constitutive expression of thednRARα. This blocks RA signalling in developing thymocytes from the DN3/4 stageonwards and thus allows us to study the role of RA in T cell development

Thymus colonisation and thymocyte positioning are regulated by interactions between CCR7 and CCR9, and their respective ligands, CCL19/CCL21 and CCL25. The ligands of CCR7 and CCR9 also interact with the atypical receptor CCRL1 (also known as ACKR4), which is expressed in the thymus and has recently been reported to play an important role in normal alpha beta T-cell development. Here, we show that CCRL1 is expressed within the thymic cortex, predominantly by MHC-II(low)CD40(-) cortical thymic epithelial cells and at the subcapsular zone by a population of podoplanin(+) thymic epithelial cells in mice. Interestingly, CCRL1 is also expressed by stromal cells which surround the pericytes of vessels at the corticomedullary junction, the site for progenitor cell entry and mature thymocyte egress from the thymus. We show that CCRL1 suppresses thymocyte progenitor entry into the thymus, however, the thymus size and cellularity are the same in adult WT and CCRL1(-/-) mice. Moreover, CCRL1(-/-) mice have no major perturbations in T-cell populations at different stages of thymic differentiation and development, and have a similar rate of thymocyte migration into the blood. Collectively, our findings argue against a major role for CCRL1 in normal thymus development and function.