Aire Controls in Trans the Production of Medullary Thymic Epithelial Cells Expressing Ly-6C/Ly-6G

Abstract

Medullary thymic epithelial cells (mTECs), which express a wide range of tissue-restricted Ags (TRAs), contribute to the establishment of self-tolerance by eliminating autoreactive T cells and/or inducing regulatory T cells. Aire controls a diverse set of TRAs within Aire-expressing cells by employing various transcriptional pathways. As Aire has a profound effect on transcriptomes of mTECs, including TRAs not only at the single-cell but also the population level, we suspected that Aire (Aire+ mTECs) might control the cellular composition of the thymic microenvironment. In this study, we confirmed that this is indeed the case by identifying a novel mTEC subset expressing Ly-6 family protein whose production was defective in Aire-deficient thymi. Reaggregated thymic organ culture experiments demonstrated that Aire did not induce the expression of Ly-6C/Ly-6G molecules from mTECs as Aire-dependent TRAs in a cell-intrinsic manner. Instead, Aire+ mTECs functioned in trans to maintain Ly-6C/Ly-6G+ mTECs. Thus, Aire not only controls TRA expression transcriptionally within the cell but also controls the overall composition of mTECs in a cell-extrinsic manner, thereby regulating the transcriptome from mTECs on a global scale.

No Major Impact of Two Homologous Proteins Ly6C1 and Ly6C2 on Immune Homeostasis

“…mTECs are classified into immature mTECs (CD80 low mTECs) and mature mTECs (CD80 high mTECs) based on the expression levels of CD80 (23,24). By using the mAbs specific for Ly6C (clone HK1.4) and Ly6G (clone 1A8), we have demonstrated that Ly6C 1 /Ly6G À , Ly6C À /Ly6G 1 , and Ly6C 1 /Ly6G 1 subsets of mature CD80 high mTECs exist with various proportions depending on the genetic background of mice (i.e., 515% in total) (21). These Ly6C-and/or Ly6G-expressing mTECs localized preferentially at the cortico-medullary junction and expressed high levels of tissue-restricted self-antigens and thymocyte-attracting chemokines.…”

“…Thymic epithelial cells (TECs) were prepared via enzymatic digestion of thymi, and subjected to flow cytometric analysis using a FACSCalibur (BD Biosciences) and a FACSAria II (BD Biosciences) as described previously (21,23,24). In brief, TECs were defined as CD45 À epithelial cell adhesion molecule 1 (EpCAM) 1 cells, and mTECs were defined as CD45 À EpCAM 1 Ulex europaeus agglutinin 1 (UEA-1) 1 cells.…”

“…In addition to monocytes/macrophages, Ly6C is expressed from other immune cells such as CD8 1 T cells (1517) and NK cells (18,19). Stromal cells including intestinal epithelial cells (20) and medullary thymic epithelial cells (mTECs) containing Aire 1 mTECs also express Ly6C (21). However, the role of the Ly6C molecules in determining the functional subsets and their possible corresponding functions remains unknown.…”

Aire suppresses CTLA-4 expression from the thymic stroma to control autoimmunity

“…Foxp3/Transcription Factor Staining Buffer Set (Invitrogen) was used for the staining of Aire, CTLA-4 and Foxp3. Flowcytometric analysis was performed using a FACSAria II (BD Biosciences) as described previously (Morimoto et al, 2018). The data were analyzed using FLOWJO 10.7.1 software (TreeStar Software) or BD FACSDiva software (version 8.0) (BD Biosciences).…”

“…Reaggregate thymic organ culture (RTOC) was performed as previously described (Morimoto et al, 2018). Briefly, thymic lobes from E15.5 to E16.5 fetuses from WT or Aire-KO were cultured on nucleopore filter (Whatman) placed in R10 containing 1.35mM 2-deoxyguanosine (2-DG) (Sigma) for 6 days.…”