*Purpose: The Autoimmune Regulator (Aire) gene, essential for central tolerance through its regulation of tissue-specific antigens in medullary thymic epithelial cells, is also expressed in extrathymic Aire-expressing cells (eTACs) in secondary lymphoid organs. eTACs can induce robust T-cell tolerance and were recently shown to be essential for the maintenance of normal maternal-fetal tolerance during pregnancy. Despite these varied roles in immune tolerance, the precise identity and biology of this population has remained elusive.

*Methods: We used transgenic Aire-reporter mice and intracellular Aire staining to identify and sort eTACs from murine lymph nodes, and subjected these populations to high-dimensional flow cytometry, scRNA-seq, and ASAP-seq. Using publicly available bulk and scRNA and ATACseq data, we defined the identity of eTACs and identified core transcriptional circuitry of the population. We used antibody-mediated RANK blockade to test its role in Aire expression in eTACs, and a mouse model of diabetes to test the ability of self-antigen expression in eTACs to prevent autoimmunity.

*Results: We defined eTACs as consisting of two distinct migratory dendritic cell-like populations, CCR7⁺ Aire-expressing migratory dendritic cells (AmDCs), and an Aire^hi population coexpressing Aire and RORγt, termed Janus cells (JCs). eTACs share transcriptional homology most closely with CCR7⁺ migratory dendritic cells, as well as medullary thymic epithelial cells. As in the thymus, RANK/RANKL signaling is required for Aire expression in eTACs, and pancreatic self-antigen expression in eTACs is sufficient to entirely prevent autoimmune diabetes in a non-obese diabetic mouse model. Intracellular Aire staining allows for the potential identification of these populations in human secondary lymphoid organs.

*Conclusions: We have defined eTACs as consisting of two distinct migratory dendritic cell-like populations, which share transcriptional and genomic homology with medullary thymic epithelial cells and can be utilized to induce T-cell tolerance and prevent autoimmunity. Validated flow-cytometric intracellular Aire staining now allows for the identification of similar populations in humans. Understanding the fundamental biology of these tolerogenic populations may have implications for a broad range of clinical applications from autoimmunity to transplantation.

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