Single-cell RNA-seq of in vitro-cultured medullary thymic epithelial cells reveals a preserved differentiation trajectory under Aire deficiency.

Central tolerance in the thymus is orchestrated by the autoimmune regulator (Aire), which enables medullary thymic epithelial cells (mTECs) to express tissue-restricted antigens (TRAs) and present them to developing T cells via MHC-II molecules. While Aire's role in mTEC heterogeneity is well established, its contribution to the transcriptome-driven differentiation trajectory remains incompletely understood. Here, we employed single-cell RNA sequencing (scRNA-seq) of in vitro-cultured murine mTECs to profile wild-type and heterozygous Aire-mutant (exon 6 c.735delG; partial loss-of-function) cells co-cultured with CD4+ thymocytes. In both genotypes, three conserved mTEC subpopulations were identified-translating, intermediate, and antigen-presentation-related. Pseudotime analysis revealed that the overall differentiation trajectory was preserved despite Aire deficiency, although with reduced transcriptional diversity, lower TRA expression, and attenuated MHC-related gene activity. Aire-mutant mTECs exhibited global transcriptional downregulation, including diminished expression of Aire partner genes involved in chromatin regulation and antigen processing. These findings indicate that Aire functions as a transcriptional amplifier and facilitator of efficient progression, enhancing the amplitude and completeness of mTEC maturation without being essential for its initiation. This study provides new insights into thymic epithelial biology and highlights the existence of Aire-independent transcriptional programs that sustain mTEC differentiation and contribute to central tolerance.