Functionally diverse thymic medullary epithelial cells interplay to direct central tolerance

Medullary thymic epithelial cells (mTECs) are essential for the establishment of self-tolerance in T cells. Promiscuous gene expression by a subpopulation of mTECs regulated by nuclear protein Aire contributes to the display of self-genomic products to newly generated T cells. Recent reports have highlighted additional self-antigen-displaying mTEC subpopulations; namely, Fezf2-expressing mTECs and a mosaic of self-mimetic mTECs including thymic tuft cells. In addition, a functionally different subset of mTECs produces chemokine CCL21 that attracts developing thymocytes to the medullary region. Here we report that CCL21+ mTECs and Aire+ mTECs non-redundantly cooperate to direct self-tolerance to prevent autoimmune pathology by optimizing the deletion of self-reactive T cells and the generation of regulatory T cells. We also detected a cooperation for self-tolerance between Aire and Fezf2, which unexpectedly regulates thymic tuft cells. Our results indicate an indispensable interplay among functionally diverse mTECs for the establishment of central self-tolerance. For SCAF1213_S_1119 and SCAF1315_S_1224, cells from the third pharyngeal region were collected. For SCAF695_190313_P5, SCAF621_P5, SCAF622_P6, SCAF696_190313_P6, SCAF804_P5 and SCAF805_P6, thymic epithelial cells from B5T-Venus embryos were isolated using FACS cell sorting according to the presence or absence of Venus signals. For SCAF2439_IRF6flox, thymic stromal cells were obtained through CD45 depletion using CD45 MicroBeads. All cells underwent analysis via Single Cell 3' end-counting gene expression (scRNA-Seq) using the 10X Genomics platform.