Biologically indeterminate yet ordered promiscuous gene expression in single medullary thymic epithelial cells

To induce central T‐cell tolerance, medullary thymic epithelial cells (mTEC) collectively express most protein‐coding genes, thereby presenting an extensive library of tissue‐restricted antigens (TRAs). To resolve mTEC diversity and whether promiscuous gene expression (PGE) is stochastic or coordinated, we sequenced transcriptomes of 6,894 single mTEC, enriching for 1,795 rare cells expressing either of two TRAs, TSPAN8 or GP2. Transcriptional heterogeneity allowed partitioning of mTEC into 15 reproducible subpopulations representing distinct maturational trajectories, stages and subtypes, including novel mTEC subsets, such as chemokine‐expressing and ciliated TEC, which warrant further characterisation. Unexpectedly, 50 modules of genes were robustly defined each showing patterns of co‐expression within individual cells, which were mainly not explicable by chromosomal location, biological pathway or tissue specificity. Further, TSPAN8+ and GP2+ mTEC were randomly dispersed within thymic medullary islands. Consequently, these data support observations that PGE exhibits ordered co‐expression, although mechanisms underlying this instruction remain biologically indeterminate. Ordered co‐expression and random spatial distribution of a diverse range of TRAs likely enhance their presentation and encounter with passing thymocytes, while maintaining mTEC identity.