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-MHCI or MHCII by the T cell antigen receptor (TCR) determines the CD8+ T cell 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 state for each T cell lineage. These analyses identified a sequential process whereby all thymocytes initiate CD4+ T cell lineage differentiation during an initial 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 multiple redundant mechanisms in guiding lineage choice. CITE-seq with a panel of 111 antibodies was performed on cells from the thymus of wild-type and lineage-restricted mice at age 4-8 weeks. Each of 19 samples was processed in a separate lane of 10x Chromium. Samples included six lineage-restricted mouse genotypes (two biological replicates each) and wild-type mice (five biological replicates processed in seven samples). 72,042 cells were captured across all samples.