Aire and Fezf2 Shape the Medullary Thymic Epithelial Cell Immunopeptidome for Central Tolerance

Medullary thymic epithelial cells (mTECs) are central to immune self-tolerance owing to their capacity to express and present a diverse repertoire of self-derived peptides to developing thymocytes. This diversity arises from both promiscuous gene expression and specialised differentiation programmes, including thymic mimetic-cell populations, and is largely shaped by AIRE/Aire and FEZF2/Fezf2. Although the roles of Aire and Fezf2 in regulating peripheral tissue antigen transcription are well established, a critical conceptual gap remains regarding how transcriptional promiscuity is translated into the repertoire of peptides presented by major histocompatibility complex class II (MHC-II) molecules. In this review and perspective, we synthesize knowledge on mTEC biology, Aire- and Fezf2-dependent transcriptional programmes, and advances in immunopeptidomics. We discuss regulatory layers that decouple mRNA abundance from peptide presentation and highlight how single-cell transcriptomics and mass spectrometry-based immunopeptidomics provide complementary insights into mTEC heterogeneity and antigenic output. We argue that integrating these approaches is essential to understand central tolerance mechanistically and to explain selective defects in thymic self-antigen presentation. This framework offers a refined basis for interpreting autoimmune disease origins and guiding antigen-focused therapeutic strategies.