Expression of tissue-restricted self-antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for self-tolerance induction and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA-sequencing and provide evidence for numerous recurring TRA co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process, which might involve local re-modeling of chromatin and thus ensures a comprehensive representation of the immunological self.

The basic two-step terminal differentiation model of the medullary thymic epithelial cell (mTEC) lineage from immature MHC class II (MHCII) to mature MHCII mTECs has recently been extended to include a third stage, namely the post-Aire MHCII subset as identified by lineage-tracing models. However, a suitable surface marker distinguishing the phenotypically overlapping pre- from the post-Aire MHCII stage has been lacking. In this study, we introduce the lectin agglutinin (TPA) as a novel cell surface marker that allows for such delineation. Based on our data, we derived the following sequence of mTEC differentiation: TPAMHCII → TPAMHCII → TPAMHCII → TPAMHCII Surprisingly, in the steady-state postnatal thymus TPAMHCII pre-Aire rather than terminally differentiated post-Aire TPAMHCII mTECs were marked for apoptosis at an exceptionally high rate of ∼70%. Hence, only the minor cycling fraction of the MHCII subset (<20%) potentially qualified as mTEC precursors. FoxN1 expression inversely correlated with the fraction of slow cycling and apoptotic cells within the four TPA subsets. TPA also further subdivided human mTECs, although with different subset distribution. Our revised road map emphazises close parallels of terminal mTEC development with that of skin, undergoing an alternative route of cell death, namely cornification rather than apoptosis. The high rate of apoptosis in pre-Aire MHCII mTECs points to a "quality control" step during early mTEC differentiation.

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