Single-cell RNA sequencing reveals regulatory patterns of self-antigen expression by medullary thymic epithelial cells (BA8P.162)

The crucial capability of T cells for discrimination between self and non-self peptides is based on rigorous negative selection of developing thymocytes by medullary thymic epithelial cells (mTECs). The mTECs purge autoreactive T cells by apparently stochastic expression of cell-type specific genes referred to as tissue-restricted antigens (TRAs). The expression of many TRAs is promoted by the autoimmune regulator (AIRE) protein, while others are induced by yet unknown factors. Despite its tremendous immunological significance, the regulation of TRA expression in mTECs is incompletely understood. Recently developed single-cell RNA-sequencing enables systematic dissection of such genome-wide patterns for the first time. Here we present analysis of 152 murine mTEC transcriptomes, revealing a regulatory hierarchy within the highly diverse population of TRA genes. We find that TRAs dependent on AIRE exhibit distinct transcriptional dynamics, with high degree of genomic clustering and characteristic transcriptional burst profiles. We show that expression of other, AIRE-independent TRAs, is likely to be secured by regulatory pathways promoting differentiation of the mTEC cells from the TEC progenitors. The TRA repertoire is further shaped by the maturation of the mTECs to MHCII high state. We further show that certain subsets of TRAs are neglected by the mTEC population, and that mTEC-driven negative selection of thymocytes may have intrinsic leakiness.