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

We show that thymic epithelial cells in the medulla (mTECs) that present self-antigens (self-Ags) to developing thymocytes for establishing immunological self-tolerance also express CTLA-4 if Aire, loss of which is responsible for the hereditary autoimmunity, is nonfunctional. Upon binding with its ligand, CD80 and CD86, expressed on thymic DCs, CTLA-4/ligand complex was internalized by Aire-deficient mTECs. This attenuated the ability of DCs to provide costimulatory signals and to present self-Ags, resulting in the reduced production of Tregs. FACS-sorted CD45-EpCAM+ TECs from Aire-KO at eight weeks of age were prepared for scRNA-seq analyses. In brief, cell suspensions were loaded onto a Chromium instrument (10× Genomics) to generate a single‑cell emulsion. scRNA-seq libraries were prepared using Chromium Single Cell 3′ Reagent Kits v3 Chemistry and sequenced in multiplex on the Illumina NovaSeq platform. FASTQ files were processed using Fastp, and reads were demultiplexed and mapped to the mm10 reference genome via Cell Ranger (v3.0.0). Processing data by the Cell Ranger pipeline was performed using the HOKUSAI supercomputer at RIKEN and the NIG supercomputer at ROIS National Institute of Genetics. Expression count matrices were prepared by counting unique molecule identifiers.