PROJECT SUMMARY/ABSTRACT The autoimmune regulator (Aire) gene, a key transcriptional regulator expressed in medullary thymic epithelial cells (mTECs), has been shown to be crucial for central tolerance by inducing tissue specific antigen (TSA) expression in mTECs. Interestingly, Aire is also found in extrathymic Aire-expressing cells (eTACs) in the secondary lymphoid organs such as the spleen and lymph nodes. We previously found that eTACs are hematopoietic antigen-presenting cells (APCs) and consist of two similar cell types: CCR7+ Aire-expressing migratory dendritic cells (AmDCs) and an Aire-high population co-expressing Aire and retinoic acid receptor– related orphan receptor γt (ROR γt) that we termed Janus cells (JCs). Functionally, eTACs are capable of enforcing deletion and anergy on self-reactive T cells, and self-antigen expression in eTACs is sufficient to prevent autoimmunity. The transcriptional, genomic, and functional symmetry between eTACs (both JCs and AmDCs) and mTECs potentially identifies a core program driven by Aire that may influence self-representation and tolerance across the spectrum of immune development. However, the lineage relationship of eTACs, what role Aire plays in these populations, and how extrathymic Aire and eTAC subsets contribute to immune homeostasis are still unknown. This proposal will test the hypothesis that Aire is inducing a tolerogenic phenotype in eTACs and that extrathymic Aire and eTACs are important for enforcing peripheral immune tolerance. Aim 1 of this proposal will define the lineage relationship between eTACs subsets, their antigen processing and presentation functions, and their migratory abilities. Aim 2 will define the cell-intrinsic functions of Aire in eTACs at both the transcriptional and chromatin level. Aim 3 will investigate the contribution of extrathymic Aire and eTACs in maintaining normal immune homeostasis. This proposal will be carried out using a variety of methods including single cell multiomics, flow cytometry, and functional approaches such as ex vivo co-cultures and in vivo monitoring of autoimmunity utilizing novel genetic mouse models. By characterizing eTAC subsets and investigating the functional roles of extrathymic Aire and eTACs, this work will help further elucidate the function of Aire and define basic peripheral tolerance mechanisms. Furthermore, understanding the biology of these tolerogenic populations may have significance for a range of clinical applications from autoimmunity to tumor immunity to maternal-fetal tolerance. This research project and fellowship training will be conducted at a top-funded research institution, the University of California, San Francisco (UCSF), in the laboratories of Dr. James Gardner and Dr. Mark Anderson. Dr. Gardner has expertise in studying peripheral Aire/eTACs and the generation of genetic mouse models. Dr. Anderson is a world expert on Aire biology, thymic selection, and immune tolerance, and a highly respected mentor and...