Aire-expressing thymic medullary epithelial cells originate from β5t-expressing progenitor cells

Abstract

The thymus provides multiple microenvironments that are essential for the development and repertoire selection of T lymphocytes. The thymic cortex induces the generation and positive selection of T lymphocytes, whereas the thymic medulla establishes self-tolerance among the positively selected T lymphocytes. Cortical thymic epithelial cells (cTECs) and medullary TECs (mTECs) constitute the major stromal cells that structurally form and functionally characterize the cortex and the medulla, respectively. cTECs and mTECs are both derived from the endodermal epithelium of the third pharyngeal pouch. However, the molecular and cellular characteristics of the progenitor cells for the distinct TEC lineages are unclear. Here we report the preparation and characterization of mice that express the recombinase Cre instead of β5t, a proteasome subunit that is abundant in cTECs and not detected in other cell types, including mTECs. By crossing β5t-Cre knock-in mice with loxP-dependent GFP reporter mice, we found that β5t-Cre-mediated recombination occurs specifically in TECs but not in any other cell types in the mouse. Surprisingly, in addition to cTECs, β5t-Cre-loxP-mediated GFP expression was detected in almost all mTECs. These results indicate that the majority of mTECs, including autoimmune regulator-expressing mTECs, are derived from β5t-expressing progenitor cells.T hymic epithelial cells (TECs) are derived from the endodermal epithelium of the third pharyngeal pouch (1-3). Cortical TECs (cTECs) provide a microenvironment that induces the generation of T cells and the positive selection of functionally competent T cells, whereas medullary TECs (mTECs) essentially contribute to the establishment of self-tolerance by the deletion of self-reactive T cells and the generation of regulatory T cells (4, 5). The nuclear protein Autoimmune regulator (Aire) expressed by a subpopulation of mTECs is essential, especially during the perinatal period, for the establishment of selftolerance in T cells (6, 7). Although the importance of the forkhead transcription factor Foxn1 for the development of both cTECs and mTECs has been established (2, 8), it remains unknown how the endodermal epithelium of the third pharyngeal pouch gives rise to cTECs and mTECs. In particular, the molecular and cellular mechanisms underlying the separate development of the cTEC and mTEC lineages remain unclear.We previously reported β5t, a proteasome subunit expressed in cTECs (9, 10). β5t is pivotal for the positive selection of immunocompetent CD8 + T cells (11,12). β5t mRNA and protein are prominently expressed in cTECs and not detected in other cell types, including mTECs (10). To examine whether β5t-expressing cells could contribute to the development of cells other than cTECs, we engineered mice in which the β5t-encoding genomic sequence was replaced with the sequence that encodes the loxP-specific recombinase Cre. Analyzing mice that are crossed to carry the β5t-Cre knock-in allele and the loxPdependent GFP reporter allele, we demonstrate that β5t-Cr...

CCRL1 marks heterogeneity in cortical and medullary thymic epithelial cells

“…-2875 that CCRL1 + fetal cTECs contain mTEC progenitor activity. These results agree with the recent reports discussed above showing that pTECs progress through a stage in which they express cTECassociated molecules before diversifying into mTECs [11,[14][15][16] (Fig. 1).…”

“…In those mice, EGFP expression would indicate current and/or past expression of β5t in the cells. It was found that β5t-Cremediated EGFP expression was detectable in almost all mTECs, including the Aire + subpopulation, throughout the ontogeny, indicating that a majority of mTECs, in which β5t expression is not detectable, are derived from β5t-expressing progenitors [16]. Collectively, the three independent reports [14][15][16] revealed a new stage in the TEC developmental pathways, i.e.…”

“…Revealing the mechanisms that underlie the specific expression of these molecules among the different TEC subpopulations would be useful to understand the molecular and cellular mechanisms for the diversification of TEC subpopulations. Along these lines, recent experiments have shown that mTECs are derived from progenitors that express cTEC-associated molecules [14][15][16]. Baik et al showed that the expression of CD205 in a fraction of TECs was detectable in embryonic day 11 (E11) mouse embryos ([14]; commented on in [17]).…”

“…Development and heterogeneity of cTECs and mTECs. pTECs progress through a stage in which they express cTEC-associated molecules (cpTECs; cTEC-like progenitor TECs), including CCRL1 CD205, β5t, and high levels of IL7, before diversifying into mTECs [11,[14][15][16][17][18]. Signals provided by developing thymocytes regulate the development of CCRL1 high CD205 + β5t + IL7 high cTECs.…”

“…Using transgenic mice that expressed yellow fluorescence protein (YFP) under the control of the IL-7 promoter sequence, in which YFP + cells represented cells that highly expressed IL-7, Ribeiro et al in a paper published in 2013 showed that YFP + TECs isolated from E14.5 mice gave rise to CD80 + mTECs in RTOCs, indicating that embryonic TECs that express high levels of IL-7, and so resemble cTECs, retain the potential to differentiate into mTECs [15]. Ohigashi et al engineered mice so that the coding sequence of the thymoproteasome β5t gene was replaced with the loxP-specific recombinase Cre, and crossed those mice with CAG-loxP-stop-loxP-EGFPtransgenic reporter mice, in which EGFP would be ubiquitously expressed under the control of the CAG promoter only when the loxP-flanked stop sequences were excised by Cre expression [16]. In those mice, EGFP expression would indicate current and/or past expression of β5t in the cells.…”