Aire suppresses CTLA-4 expression from thymic stroma to control autoimmunity. 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. Overall design: 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.

本研究证实，在建立免疫自身耐受过程中，向发育中胸腺细胞呈递自身抗原（self-antigens, self-Ags）的胸腺髓质上皮细胞（medullary thymic epithelial cells, mTECs），在自身免疫调节因子（Autoimmune Regulator, Aire）功能丧失时也会表达细胞毒性T淋巴细胞相关蛋白4（CTLA-4）——而Aire的缺失正是遗传性自身免疫病的致病原因。当CTLA-4与胸腺树突状细胞（thymic dendritic cells, DCs）表面表达的配体CD80和CD86结合后，CTLA-4-配体复合物会被Aire缺陷型mTECs内吞。这会削弱DCs提供共刺激信号以及呈递自身抗原的能力，最终导致调节性T细胞（regulatory T cells, Tregs）的生成量减少。

实验整体设计：选取8周龄Aire基因敲除（Aire-KO）小鼠，通过流式细胞术分选（fluorescence-activated cell sorting, FACS）得到CD45⁻EpCAM⁺胸腺上皮细胞（thymic epithelial cells, TECs），用于单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析。简要而言，将细胞悬液加载至Chromium系统（10× Genomics）以制备单细胞油包水乳液。scRNA-seq文库使用Chromium Single Cell 3′ Reagent Kits v3 Chemistry构建，并在Illumina NovaSeq平台上进行多重测序。使用Fastp处理FASTQ文件，通过Cell Ranger（v3.0.0）对测序reads进行解多重比对，并将其映射至mm10参考基因组。Cell Ranger数据分析流程的运行依托日本理化学研究所（RIKEN）的HOKUSAI超级计算机与ROIS国立遗传学研究所（NIG）的NIG超级计算机完成。通过统计唯一分子标识符（unique molecule identifiers, UMIs）的数量，生成基因表达计数矩阵。