During thymic development, thymocytes adjust their TCR response based on the strength of their reactivity to self-peptide MHC complexes. This tuning process allows thymocytes with a range of self-reactivities to survive positive selection and contribute to a diverse T cell pool. In this review, we will discuss recent advances in our understanding of how thymocytes tune their responsiveness during positive selection, and we present a sequential selection model to explain how MHC specificity influences lineage choice. We also discuss recent evidence for cell type diversity in the medulla and discuss how this heterogeneity may contribute to medullary niches for negative selection and regulatory T cell development.

The development of CD4+ T cells and CD8+ T cells in the thymus is critical to adaptive immunity and is widely studied as a model of lineage commitment. Recognition of self-peptide major histocompatibility complex (MHC) class I or II by the T cell antigen receptor (TCR) determines the CD8+ or CD4+ T cell lineage choice, respectively, but how distinct TCR signals drive transcriptional programs of lineage commitment remains largely unknown. Here we applied CITE-seq to measure RNA and surface proteins in thymocytes from wild-type and T cell lineage-restricted mice to generate a comprehensive timeline of cell states for each T cell lineage. These analyses identified a sequential process whereby all thymocytes initiate CD4+ T cell lineage differentiation during a first wave of TCR signaling, followed by a second TCR signaling wave that coincides with CD8+ T cell lineage specification. CITE-seq and pharmaceutical inhibition experiments implicated a TCR-calcineurin-NFAT-GATA3 axis in driving the CD4+ T cell fate. Our data provide a resource for understanding cell fate decisions and implicate a sequential selection process in guiding lineage choice.