T cell tolerance depends heavily on the thymus, reflecting negative selection of effector T cells (Teffs) and positive selection of regulatory T cells (Tregs). Medullary thymic epithelial cells (mTECs) are major players in both of these processes. Beyond transcribing the genes encoding molecules needed for their maintenance and function, mTECs express transcripts derived from much of the protein-coding genome, notably mRNAs specifying proteins characteristic of mature peripheral cell-types (peripheral-tissue antigens or PTAs). While the whole mTEC compartment expresses a large repertoire of PTA transcripts, an individual mTEC makes only a small subset of them. Aire drives expression of most thymic PTA transcripts by a mechanism thought to entail super-enhancer activation and remodeling of 3D chromatin structure. Hence, the reigning model to account for the mTEC PTA repertoire focuses on quasi-random de-repression of gene transcription, with no discernible biological logic. However, our unpublished scATAC-seq and multi-pronged downstream studies argue for a different model: in fact, Aire promotes both the ectopic expression of PTA transcripts (in Aire-stage mTECs) and the ectopic differentiation of peripheral cell-types (from post-Aire mTECs). While previous scRNA- seq studies did uncover some ectopic mTEC clusters, our ATAC-seq approach substantially broadened these observations and revealed the underlying logic: a diversity of ectopic mTEC subtypes, largely downstream of Aire expression, are marked by the lineage-defining transcription factors (TFs), chromatin accessibility landscapes and transcriptional programs of peripheral cell-types. While it is well established that the ectopic transcripts characteristic of Aire-expressing mTECs play an important role in T cell tolerance, almost nothing is known about the influence of post-Aire ectopic mTEC subtypes. Thus, the major goal of this project is to elucidate the impact of ectopic mTEC subtypes on T cell tolerance, which will be addressed in three Specific Aims: a. To determine whether ectopic mTEC subtypes induce T cell tolerance. Our initial approach relies on the lineage-defining TF logic mentioned above and on exploiting a fluorescent-protein (FP)-targeting strategy. Preliminary data demonstrate that specific targeting of eYFP to the M-cell mTEC subtype inhibits the CD4+ T cell response to immunization with an eYFP peptide. We will extend this finding in several directions -- evaluating the mechanism, thymus dependence, Aire dependence, generality & implications for autoimmunity. b. To examine the landscape and function of ectopic mTEC subtypes in perinatal mice. As this is a critical window for induction of T cell tolerance, we will perform scRNA-seq, scATAC-seq and functional comparisons of mTECs from perinatal and adult mice to query the range and relative importance of ectopic mTEC subtypes at the two ages. c. To determine whether extrinsic signaling to ectopic mTEC subtypes expands their...