Single-cell multiomics defines tolerogenic extrathymic Aire-expressing populations with unique homology to thymic epithelium

The autoimmune regulator (Aire), a well-defined transcriptional regulator in the thymus, is also found in extrathymic Aire-expressing cells (eTACs) in the secondary lymphoid organs. eTACs are hematopoietic antigen-presenting cells and inducers of immune tolerance, but their precise identity has remained unclear. Here, we use single-cell multiomics, transgenic murine models, and functional approaches to define eTACs at the transcriptional, genomic, and proteomic level. We find that eTACs consist of two similar cell types: CCR7(+) Aire-expressing migratory dendritic cells (AmDCs) and an Aire(hi) population coexpressing Aire and retinoic acid receptor-related orphan receptor gamma t (ROR gamma t) that we term Janus cells (JCs). Both JCs and AmDCs have the highest transcriptional and genomic homology to CCR7(+) migratory dendritic cells. eTACs, particularly JCs, have highly accessible chromatin and broad gene expression, including a range of tissue-specific antigens, as well as remarkable homology to medullary thymic epithelium and RANK-dependent Aire expression. Transgenic self-antigen expression by eTACs is sufficient to induce negative selection and prevent autoimmune diabetes. This transcriptional, genomic, and functional symmetry between eTACs (both JCs and AmDCs) and medullary thymic epithelium-the other principal Aire-expressing population and a key regulator of central tolerance-identifies a core program that may influence self-representation and tolerance across the spectrum of immune development.