scRNA-seq of mouse thymocyte CD4 CD8 lineage choice and differentiation

CD4 and CD8 identify helper and cytotoxic T cell lineages, and serve as co-receptors for MHC-restricted TCR recognition. Despite decades of investigation, questions remain about how TCR specificity and co-receptor gene expression are matched during T cell repertoire selection. Here we address the order and logic of CD4/CD8 lineage choice and differentiation by scRNA-seq. Maturation, activation, and lineage specification emerged as principle components, in this order. Co-receptor gene expression uncovered waves of Cd4+Cd8a+, Cd4+Cd8a- and Cd4-Cd8a+ selection intermediates and revealed gene expression programs associated with each pattern, including TCR- and cytokine signaling genes, lineage markers and transcription factors. While in the unperturbed thymus early Cd4+Cd8a+ selection intermediates were followed first by CD4-like Cd4+Cd8a- and later by CD8-like Cd4-Cd8a+ selection intermediates, the order of co-receptor gene expression was not inherently fixed. These data provide a benchmark for models of CD4/CD8 lineage choice, and a rich resource for interrogating T cell differentiation. Overall design: Thymocyte cell suspensions from two wild type C57BL/6 thymi and one MHC class II-deficient thymus (Madsen et al., Proc Natl Acad Sci U S A. 1999; 96: 10338-43) were stained with antibodies to CD4, CD8a, TCRb and CD69-. Single cells representing thymocytes before, during and after lineage choice and differentiation (CD4+ CD8+ Cd69, CD4+ CD8+ Cd69+, CD4+ CD8+ TCRbhi , CD4+ CD8low, CD4+ CD8 -, and CD4- CD8+ TCRbhi) were sorted by flow cytometry into 96 well plates containing lysis buffer for construction of RNA-seq libraries.