Abstract The Autoimmune Regulator ( Aire ) gene, well defined for its role in medullary thymic epithelial cells (mTECs) and immune self-tolerance, is also expressed in extrathymic Aire -expressing cells (eTACs) in the secondary lymphoid organs. eTACs have been shown to be hematopoietic antigen presenting cells (APCs) and potent inducers of immune tolerance (1–3). However, the precise identity and function of these cells remain unclear. Here, we use high-dimensional single-cell multiomics 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 a unique Aire-hi population co-expressing Aire and RAR-related orphan receptor gamma-t (RORγt). The latter, which have significant transcriptional and genomic homology to migratory dendritic cells (migDCs) and mTECs, we term Janus cells (JCs). All eTACs, and JCs in particular, have a highly accessible chromatin structure and high levels of broad gene expression, including tissue-specific antigens, as well as remarkable transcriptional and genomic homology to thymic medullary epithelium. As in the thymus, Aire expression in eTACs is also dependent on RANK-RANK-ligand interactions. Furthermore, lineage-tracing shows that JCs are not precursors to the majority of AmDCs. Finally, self-antigen expression by eTACs is sufficient to mediate negative selection of T cells escaping thymic selection and can prevent autoimmune diabetes in non-obese diabetic mice. This transcriptional, genomic, and functional symmetry between a hematopoietic Aire-expressing population in the periphery and an epithelial Aire-expressing population in the thymus suggests that a core biological program may influence self-tolerance and self-representation across the spectrum of immune development.

Single-Cell Multiomics Defines Tolerogenic Extrathymic Aire-Expressing Populations with Unique Homology to Thymic Epithelium