Single-cell multiomic analysis of thymocyte development reveals drivers of CD4⁺ T cell and CD8⁺ T cell lineage commitment

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.